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BIOLOGY 
LBMIARY 


A  LABORATORY  GUIDE  IN  ELEMENTARY 
BACTERIOLOGY 


A  LABORATORY  GUIDE 


IN 


Elementary  Bacteriology 


BY 


WILLIAM   DODGE  FROST.  PH.  D 

\\ 
Associate  Professor  of  Bacteriology,  University  of  Wisconsin 


FIFTH    REVISED    EDITION 


got* 

THE  MACMILLAN  COMPANY 

LONDOV :    MACMILLAN  &  CO.,  LTD. 
1918 

Ml  Rights  Rfitried 


111? 


COPYRIGHT,  1901,  1902  AND  1904, 
BY   WILLIAM   DODGE   FROST. 


Set  up  and  clectrotyped.  Published  October,  1904.  Reprinted 
September,  1907;  July,  1909;  April,  1911;  July,  1912;  July,  1913; 
February,  1916  ;  January,  1918. 


"MortoooB 
Berwick  &  Smith  Co.,  Norwood,  Mass.,  U.S.A. 


PREFACE  TO  THE  FOUETH  EDITION 


In  this  edition  minor  changes  have  been  made.  A  few  of  the 
experiments  have  been  rewritten;  some  of  the  old  methods  have 
been  replaced  by  later,  and  it  is  hoped  better,  methods. 

The  general  plan  of  the  book  remains  the  same.  Its  object,  as 
heretofore,  is  to  give  adequate  directions  for  the  performance  of 
certain  fundamental  exercises  in  bacteriology.  In  attempting  this 
two  considerations  have  been  kept  in  mind,  first,  that  in  a  rapidly 
developing  subject  it  is  important  that  the  directions  for  the 
various  exercises  be  worded  so  as  to  lend  themselves  readily  to 
changes  which  become  desirable  from  time  to  time.  With  this 
end  in  view  the  directions  have  been  divided  where  possible  into 
a  general  and  a  special  part.  The  general  directions  contain  the 
essential  part  of  the  exercise  which  does  not  permit  of  any  con- 
siderable variation,  while  the  special  directions  embrace  such 
features  as  are  most  subject  to  modification,  as  for  instance  the 
particular  organism  to  be  used,  the  kind  of  medium,  the  incuba- 
tion temperature,  etc.  Desirable  changes  here  are  easily  indi- 
cated when  the  exercise  is  assigned ;  second,  that  each  experiment 
should  be  complete  in  itself.  Thus  some  of  the  experiments  can 
be  performed  in  a  few  moments,  while  others  require  several  days 
for  their  completion.  No  attempt  has  been  made  to  group  them 
into  lessons.  The  order  of  the  experiments  is  believed  to  be  a 
logical  one,  but  may  be  readily  adapted  to  meet  the  needs  under 
varying  conditions. 

The  various  bacteria  are  studied  in  groups.  This  arrangement 
is  in  keeping  with  recent  tendencies,  and  it  is  hoped  that  it  will 
impress  the  student  with  the  similarity  between  closely  related 
forms,  and  also  emphasize  certain  minute  but  important  differ- 
ences. 

The  system  of  classification  adopted  is  that  suggested  by 
Migula  and  is  the  one  most  widely  accepted. 

The  nomenclature  used  is  determined  by  rules  generally  adopt- 
ed by  systematists.  To  those  who  still  prefer  the  old  names,  the 
synonyms  will  be  found  useful. 

(v) 


vi  PREFACE 

The  charts  of  the  various  organisms  furnish  a  most  satisfactory 
means  for  recording  the  observations  made  during  the  study  of  a 
germ  and  are  especially  convenient  for  reference. 

Blank  pages  have  been  left  for  notes  and  drawing  with  the 
idea  that  notes  in  permanent  form  are  the  only  ones  of  value  to 
the  student  in  subsequent  years. 

References  have  been  made  to  the  leading  text-books  and  occa- 
sionally to  original  sources.  It  is  expected  that  the  student  will 
make  constant  use  of  these  references. 

My  acknowledgments  are  due  to  my  colleagues,  Professor  C.  A. 
Fuller  and  Miss  Vermillion  Armstrong. 

W.  D.  FROST. 

Madison,  Wis.,  January,  1911. 


TABLE  OF  CONTENTS. 


List  of  Apparatus X 

Laboratory  Eules XI 

List  of  Keferences XII 

PAET  I.— GENERAL  BACTEEIOLOGY 
CHAPTER  I.    MORPHOLOGY  AND  ELEMENTARY  TECHNIQUE. 


EXERCISE  PAGE 

1.  Cleaning  Glassware 2 

2.  Plugging     Flasks     and 

Tubes 4 

3.  Sterilization  of  Glassware  4 

4.  Preparation  of  Bouillon...  6' 

5.  Filling      Test-tubes     and 

Flasks  with  Culture  Me- 
dia     10 

6.  Sterilization  of  Culture 

Media 10 

7.  Preparation  of  Gelatin. . .  14 

8.  Preparation  of  Agar 16 

9.  Preparation  of  Potatoes..  18 

10.  Preparation   of   Water- 

blanks 18 

11.  Care  of  Culture  Media 18 

12.  Platinum   Needles 20 

13.  Test-tube  Cultures 22 

14.  Incubation  of  Cultures. ...  24 

15.  Study    of    Test-tube    Cul- 

tures   26 

16.  Cleaning  Slides  and  Cover- 

glasses  26 

17.  Preparation     of     Staining 

Solution 28 

18.  Simple    Cover-glass    Prep- 

arations.. .  30 


EXERCISE  •    PAGE 

19.  Use  of  Microscope 34 

20.  Drawing  Bacteria 36 

21.  Hanging-drop  Prepara- 

tion 38 

22.  Microscopical    Study    of 

Form  Types 40 

23.  Study  of  Cell  Grouping.  . .  42 

24.  Study  of  Involution  Forms  46 

25.  Study  of  Endospores 46 

26.  Flagella  Stain 48 

27.  Capsule    Stain 50 

28.  Stain    for    Metachromatic 

Granules 50 

29.  Morphology  of  Yeasts  and 

Moulds    Compared    with 

Bacteria    50 

30.  Gelatin  Plate  Cultures 52 

31.  Agar   Plate   Cultures 56 

32.  Koll    Cultures 56 

33.  Study  of  Plate  Cultures...  58 

34.  Use   of   Decolorizing 

Agents 60 

35.  Gram's   Stain 60 

36.  Tubercle   Stain    (Gabbett)  62 


CHAPTER  II.    PHYSIOLOGY  OF  BACTERIA. 


EXERCISE  PAGE 

37.  Preparation  of  Special 

Media 64 

38.  Effect  of  Keaction  of  Me- 

dia on  Growth 66 

39.  Effect  of  Concentration  of 

Media  on  Growth . .  66 


EXERCISE  PAGE 

40.  Effect  of  Temperature 

Variations    on    Bate    of 
Growth    68 

41.  Determination  of  Thermal 

Death  Point..  68 


(VII) 


viii 


TABLE  OF  CONTENTS 


CHAPTER  II.     PHYSIOLOGY  OF  BACTERIA. — Continued 


42. 

43. 
44. 

45. 
46. 

47. 
48. 

49. 
50. 


Comparative  Efficiency  of 

Dry  and  Moist  Heat 70 

Effect  of  Desiccation.....     70 
Effect     of     Chemicals     on 

Bacteria 72 

Kelation  to  Oxygen 72 

Effect  of  Direct  Sunlight..      72 

Detection  of  Gas 74 

Quantitative    Analysis    of 

Gas 74 

Petection  of  Acids  and  Al- 
kalies      76 

Quantitative      Determina- 
tion of  Acids. .  76 


EXERCISE  PAGE 

51.  Detection    of    Nitrites    in 

Cultures 76 

52.  Detection  of  Ammonia...     78 

53.  Detection  of  Sulphuretted 

Hydrogen .  .      78 

54.  Detection  of  Indol 78 

55.  Determination  of  Chemical 

Enzyms  in  Cultures. ...      80 

56.  Variation    in    Enzym   Pro- 

duction       80 

57.  Variation  in  Color  Produc- 

tion. . ,  80 


CHAPTER  III.    TAXONOMY. 


Points    to    be    Observed    in    the 

Study  of  Bacteria 82 

Classification    of    Bacteria    (Mi- 

gula) 89 


PAGE 

Bacteria     Arranged    in     Classes 

and    Groups 91 


CHAPTER  IV.    SYSTEMATIC  STUDY  OF  REPRESENTATIVE  NON  PATHOGENIC 

BACTERIA. 


EXERCISE  PAGE 

58.  Preparation  of  Special 

Media 94 

59.  Saprophilic   Class 95 

60.  Chromogenic    Class 103 


EXERCISE  PAGE 

62.  Separation     of     Bacterial 

Coloring  Matter 108 

63.  Zymogenic    Class 109 

64.  Saprogenic  Class 113 

65.  Phosphorescent  Class 121 


61.     Variety  of  Pigments 107 

CHAPTER  V.    BACTERIOLOGICAL  ANALYSIS. 


EXERCISE  PAGE 

66.  Comparative    Analysis    of 

Air 126 

67.  Quantitative      Determina- 

tion of  Number  of  Bac- 
teria in  Air 126 

Water   Analysis 128 

Estimation   of  Number   of 
Bacteria   in   Soil..          .    132 


68. 
69. 


EXERCISE 

70. 


PAGE 

Quantitative    Analysis    of 
Milk 132 

71.  Efficiency     of     Pasteuriza- 

tion     132 

72.  Testing  Antiseptic  Action 

of   Chemicals 134 

73.  Testing    Disinfecting    Ac- 

tion of  Chemicals 136 


PAKT  II.— MEDICAL  BACTEEIOLOGY. 
CHAPTER  VI.    PATHOGENIC  AEROBES. 


EXERCISE  PAGE 

74.  Preparation  of  Culture 

Media 138 

75.  Erysipelas   Group 141 


EXERCISE  PAGE 

76.  Pus  Coccus  Group 145 

77.  Malta   Fever   Group 153 

78.  Diplococcus    Group 157 


TABLE  OF  CONTENTS 


ix 


CHAPTER  VI.    PATHOGENIC  AEROBES. — Continued. 


EXERCISE 


79. 

80. 
81. 
82. 
83. 
84. 
85. 
86. 


Sarcina    Group 

Anthrax    Group 

Friedlander  Group . . 
Swine  Plague  Group. 

Glanders   Group 

Diphtheria  Group. .  . 
Pneumonia  Group... 
Influenza  Group 


PAGE          EXERCISE 
163 

167 
171 
183 
191 
199 
207 
211 


89. 
90. 
91. 
92. 
93. 


Tubercle   Group 213 

Colon  Group 219 

Hog  Cholera  Group 227 

Typhoid    Group 235 

Pseudomonas   Group 247 

Cholera    Group 251 

Streptothrix    Group 263 


CHAPTER  VII.     PATHOGENIC  ANAEROBES 


EXERCISE  PAGE 

94.  Emphysema    Group 273 

95.  Oedema  Group 277 


EXERCISE 

96.     Tetanus  -Group. 


PAGE 

,   289 


CHAPTER  VIII.     ANIMAL  INOCULATION  AND  STAINING  OF  BACTERIA  IN  TISSUE. 


EXERCISE  PAGE 

97.  Animal   Inoculation 294 

98.  Preparation  of  Tissue  for 

Examination. . .  .   310 


EXERCISE  PAGE 

99.     Staining    Sections. 312 


CHAPTER  IX.    BACTERIOLOGICAL  DIAGNOSIS. 


EXORCISE  PAGE 

100.  Examination      of     Buccal 

Secretion 320 

101.  Examination  of  Sputum..   326 

Examination  of  Blood 330 

Examination  of  Feces 338 

Examination  of  Urine .  .    .   346 


102. 
103. 
104. 


PAGE 


EXERCISE 

105.  Examination     of     Transu- 

dates  and  Exudates.  . . .   346— 

106.  Diagnosis  of  Rabies 354 

107.  Examination    of    Material 

from    Human    Autopsies  356 


CHAPTER  X.    DETECTION  OF  PATHOGENIC  BACTERIA  IN  WATER  AND  MILK 

SUPPLIES. 


EXERCISE  PAGE 

108.     Examination  of  Water  for 

Pathogenic   Bacteria. . . .   358 


109.     Examination    of   Milk   for 


PAGE 


Pathogenic    Bacteria 360 


APPENDIX  A. 


A  Key  to  the  Indentification  of  the  Common  Pathogenic  and  a  few  of  the 

Well  Known  Saprophytic  Bacteria 361 

APPENDIX  B. 

Charts  for  the  study  of  additional  bacteria 365 

APPENDIX  C. 
Tables   389 

Index    .  .    391 


LIST  OF  APPARATUS 


This  list  comprises  the  apparatus  which  is  to  be  under  the 
exclusive  control  of  the  student  and  does  not  include  the  general 
laboratory  outfit,  such  as  sterilizers,  incubators,  microscopes,  gen- 
eral chemical  supplies,  etc. 


A. 

50  (%  oz.)  cover-glasses,  18  mm.  (% 
in.)    square   and   0.17   mm.    thick 
(No.  2). 
50  glass  slides. 
100  labels,  2  cm.  square. 
12  cm.   platinum  wire    (No.   27). 
1  pair    cover-glass   forceps    (Cornet 
or  Stewart). 

1  pair   fine  pointed   forceps. 

2  slide  boxes  for  50  slides. 
1  hanging-drop   slide. 

1  towel. 

1  yard  of  muslin. 

B. 

1  flask,  1000  cc. 

4  flasks,  400  cc. 

1  flask,   250  cc. 

1  flask,  100  cc. 

200  test-tubes   (15x120  mm.). 
15  Petri  dishes    (10  cm.). 
6  fermentation  tubes. 
6  glass  tumblers  or  tin  cups. 

3  small  wire  baskets. 

2  glass  rods  for  platinum  needles. 


3  pipettes,  1  cc. 

1  brass   tube   to    hold   pipettes    (25 

X   250  mm.). 
8  stain    bottles    with    pipettes,    in 

block. 

3  sheets  of  filter  paper. 
3  sheets  of  lens  paper. 
1  test-tube  brush. 
1  glass  funnel,  12  cm. 

1  glass  funnel,  5  cm. 

2  stirring  rods. 
1  pipette,  5  cc. 

1  thermometer,  0-100°  C. 
10  cm.  rubber  tubing,  1  cm.  dia. 

See  Fig.  1. 
1  Mohr  stopcock. 
1  potato  knife. 

1  Bunsen  burner  with  tubing. 
1  piece  of  wire  gauze. 
1  rice  cooker. 

1  graduated  cylinder,  300  cc. 
I  graduated   cylinder,    100   cc. 
1  graduated  cylinder,   25  cc. 
1  evaporating  dish,   10  cm. 
1  disinfecting   jar. 
1  copper  cup. 
1  ring  stand  with  clamp. 


LABORATORY  RULES 


I.  Before  beginning  an  exercise  read  over  the  directions  and  look  up 
some  of  the  references.    Keep  notes  of  everything  done  and  the  conclusions 
reached  on  the  right  hand  pages  in  this  Guide.    Make  drawings  wherever 
they  will  be  of  value.     Outline  with  pencil  and  fill  in  with  India  ink.     The 
Laboratory  Guide  should  be  kept  in  the  laboratory. 

II.  All  possible  cleanliness  should  be  observed  in  the  care  of  apparatus, 
desk,  etc. 

III.  After  working  with  the  pathogenic  bacteria  the  tables  should  be 
washed  with  corrosive  sublimate  and  the  hands  disinfected  by  washing  in 
the  sublimate  solution  (or  a  germicidal  soap)  and  then  in  soap  and  water. 

IV.  Solid  material,  culture  media  and  corrosive  sublimate  should  not 
be  put  in  the  sink  but  in  crocks  provided  for  the  purpose.     Burnt  matches, 
pieces  of  paper,  etc.,  should  also  be  put  in  the  crocks  and  not  on  the  floor. 

V.  When  using  the  steam  sterilizer  see  that  there  is  enough  water  pres- 
ent before  lighting  the  gas  and  do  not  leave  the  laboratory  until  the  gas  has 
been  turned  off. 

VI.  Food  should  not  be  eaten  in  the  laboratory  and  lead  pencils  or  labels 
should  not  be  moistened  with  the  tongue. 

VII.  All  cultures  of  bacteria  should  be  labeled  with  the  name  of  the 
organism,  the  name  of  the  student  and  the  date. 

VIII.  The  platinum  needles  used  in  making  cultures  should  be  sterilized 
shortly  before  and  immediately  after  use,  and  before  they  are  laid  down. 
When  the  needles  are  covered  with  infectious  material  they  should  be  held 
at  the  side  of  the  flame  until  dry  before  being  sterilized;  this  will  avoid  the 
danger  of  scattering  this  material  about  the  laboratory. 

IX.  Discarded  cultures  should  be  covered  with  corrosive  sublimate  and 
placed  in  a  proper  receptacle,  and  under  no  condition  should  they  be  left 
lying  about  the  laboratory.     Pipettes  which  have  been  used  to  handle  infec- 
tious material  should  be  placed  in  a  glass  cylinder  containing  a  disinfectant, 
or  potassium  bichromate  and  sulphuric  acid. 

X.  If  infectious  matter  should  by  accident  come  in  contact  with  the 
hands,  or  be  dropped  on  the  table  or  floor,  corrosive  sublimate  (1:1000)  should 
be  immediately  applied. 


(XI) 


LIST  OF  TEXTS  AND  REFERENCE  WORKS  WITH 
ABBREVIATIONS  USED 


A. —  Abbott:   Principles  of  Bacteriology.     Lea  Bros.  &  Co.,   Philadelphia,   5th 

Edit.,  1899. 
A.  2. —      Abbott :  Hygiene  of  Transmissable  Diseases.     Saunders  &  Co.,  Philadelphia, 

2nd  Edit.,  1903. 
B. —  Bowhill  :   Manual  of  Bacteriological   Technique.     Oliver  &  Boyd,   London, 

2nd  Edit.,  1902. 
C. —  Chester:  A  Manual  of  Determinative  Bacteriology.     The  Macmillan  Co., 

New  York,  1901. 
Cn. —          Conn :    Agricultural    Bacteriology.     Blakiston's   Son   &   Co.,    Philadelphia, 

1901. 

Cn.  2—      Conn  :  Bacteria  in  Milk.     Blakiston's  Son  &  Co.,  1903. 

Co. —          Connell :   A   Laboratory   Guide   in   Practical   Bacteriology.     Author,   King- 
ston, Ontario,  1899. 
Cu. —          Curtis :    Essentials   of   Practical    Bacteriology.     Longmans,    Green   &   Co., 

New  York,  1900. 
E. —  Emery  :   Handbook  of  Bacteriological  Diagnosis.     Blakiston's   Son  &  Co., 

Philadelphia,  1902. 

Ey. —          Eyre  :  Bacteriological  Technique.     Saunders  &  Co.,  Philadelphia,  1903. 
P. —  Fischer :    Structure   and   Functions   of   Bacteria.     Clarendon    Press,    New 

York,  1900. 

Fl. —  Fluegge  :  Die  Mikro-organismen.     F.  C.  W.  Vogel,  Leipzig,  1896. 

Fr. —  Frankland :    Micro-organisms    of    Water.     Longmans,    Green    &   Co.,    New 

York,  1894. 

G. —  Gage  :  The  Microscope.     Comstock  Pub.  Co.,  Ithaca,  N.  Y.,  8th  Edit.,  1901. 

Go. —          Gorham :    Laboratory    Course    in    Bacteriology.     W.    B.    Saunders    &   Co., 

Philadelphia,  1901. 
H. —  Hewlett :   Manual  of  Bacteriology.     Blakiston's   Son  &  Co.,   Philadelphia, 

2nd  Edit.,  1902. 
Ho. —          Horrocks :     Introduction    to    the   Bacteriological    Examination    of   Water. 

Blakiston's  Son  &  Co.,  Philadelphia,  1902. 
J.  H. —       Jordan's  Translation  of  Hueppe  :  Principles  of  Bacteriology.     Open  Court 

fc  Pub.  Co.,  Chicago,  1899. 

v.  J. —        v.  Jaksch  :  Clinical  Diagnosis.     Charles  Griffin  &  Co.,  London,  4th  Edit.. 

1899. 
K. —  Kloecker:    Fermentation   Micro-organisms.  Longmans,    Green   &   Co.,   New 

York,  1903. 
K.  &  D. —  Kanthack  &  Drysdale  :    Practical   Bacteriology.     The  Macmillan   Co.,   New 

York,   1895. 
K.  &  W. — Kolle  &  Wassermann  :  Handbuch  der  Pathogenen  Mikro-organismen,  I.,  II. 

and  III.,  and  atlas.     Gustav  Fischer,  Jena,  1903. 
L. —  Lafar :   Technical   Mycology,   Vol.   I.     Lippincott  Co.,   Philadelphia,   1898. 

Vol.  II.,  Part  I.,  1903. 
L.  &  K. — Levy  &  Klemperer  :  Clinical  Bacteriology.     Saunders  &  Co.,  Philadelphia, 

1900. 

L.  &  N. —  Lehmann  &  Neumann  :  Atlas  and  Essentials  of  Bacteriology.     W.  B.  Saun- 
ders &  Co.,  Philadelphia,  1901. 
M. —  Moore  :  Laboratory  Directions  for  Beginners  in  Bacteriology.     Ginn  &  Co., 

New  York,  1900. 

(XII) 


TEXTS  AND  REFERENCE  WORKS  xiii 

Mig. —        Migula  :  System  der  Bakterien.     Gustav  Fischer,  Jena,  1900. 

M.  &  B. — Muir  &  Ritchie :  Manual  of  Bacteriology.  The  Macmillan  Co.,  New  York, 
3rd  Edit.,  edited  by  Harris,  1903. 

M.  &  W.— Mallory  &  Wright :  Pathological  Technique.  W.  B.  Saunders  &  Co.,  Phila- 
delphia, 2nd  Edit.,  1903. 

McF. —  McFarland  :  Text-Book  of  Pathogenic  Bacteria.  W.  B.  Saunders  &  Co., 
Philadelphia,  4th  Edit.,  1903. 

N. —  Novy  :  Laboratory  Work  in  Bacteriology.  Geo.  Wahr,  Ann  Arbor,  Mich., 

2nd  Edit.,  1899. 

Ne. —          Newman  :  Bacteria.     Putnam,  New  York,  2nd  Edit.,  1903. 

P. —  Park  :  Bacteriology  in  Medicine  and  Surgery.  Lea  Bros.  &  Co.,  Philadel- 

phia, 1899. 

P.  B.  C. — Proceedings  of  the  Bacteriological  Committee  from  Jour.  Amer.  Pub.  Health 
Assn.,  Vol.  XXII. 

P.  &  M. — Peamain  &  Moor  :  Applied  Bacteriology.  Baillere,  Tindall  &  Cox,  London, 
2nd  Edit. 

P.  &  W. — Prescott  &  Winslow  :  Elements  of  Water  Bacteriology.    Wiley  &  Sons,  1904. 

R. —  Roger  :  Infectious  Diseases.     Lea  Bros.  &  Co.,  Philadelphia,  1903. 

S. —  Sternberg  :  Manual  of  Bacteriology.     Wood  &  Co.,  New  York,  1893. 

S.  2. —        Sternberg  :  Immunity.     Putnam  &  Sons,  New  York,  1903. 

Si.—  Simon  :  Clinical  Diagnosis.     Lea  Bros.  &  Co.,  Philadelphia,  3rd  Edit.,  1897. 

W. —  Woodhead  :  Bacteria  and  their  Products.  Charles  Scribner  &  Sons,  New 

York,  1892. 

Wm. —  Williams  :  Manual  of  Bacteriology.  Blakiston's  Son  &  Co.,  Philadelphia, 
3rd  Edit.,  1904. 


PART  I 

GENERAL  BACTERIOLOGY 


PART  I- GENERAL  BACTERIOLOGY 
CHAPTER  I 

MORPHOLOGY  AND  ELEMENTARY 
TECHNIQUE 


EXERCISE  1.     CLEANING  GLASSWARE. 

GENERAL  DIRECTIONS.  All  glassware  to  contain  culture  media 
must  be  thoroughly  cleaned.  New  glassware  should  be  washed  in 
hot  soap-suds  (a  test-tube  brush  will  be  needed  for  the  test-tubes), 
rinsed  in  tap  water  and  then  placed  for  a  few  minutes  in  water  to 
which  about  \%  of  hydrochloric  acid  has  been  added  to  remove 
free  alkali  frequently  present  on  new  glass,  and  then  thoroughly 
rinsed  in  running  water.  It  is  then  allowed  to  drain.  Test-tubes 
and  flasks  are  best  dried  by  placing  them  on  a  drain  board  spe- 
cially prepared,  or  standing  them  mouth  down  in  a  box  with  a 
cloth  bottom  or  in  a  wire  basket. 

Glassware  containing  media  (discarded  cultures,  etc.,)  is  best 
cleaned  by  first  standing  in  water  for  some  hours,  or  by  being 
steamed  and  pouring  out  the  material  while  in  a  liquid  condition 
and  then  cleaning  as  above  with  the  exception  of  the  use  of  the 
hydrochloric  acid. 

REFERENCES.     A.  126 ;  H.  44 ;  P.  223. 

SPECIAL  DIRECTIONS.  Read  Rule  I.  Clean  as  directed  above, 
all  flasks,  test-tubes,  fermentation  tubes  and  Petri  dishes  in  your 
possession. 


GENERAL  BACTERIOLOGY 


EXERCISE  2.  PLUGGING  FLASKS  AND  TUBES. 

GENERAL  DIRECTIONS.  When  the  flasks,  test-tubes  and  fer- 
mentation tubes  are  thoroughly  dry  they  are  to  be  plugged  with 
cotton.  The  cotton  for  this  purpose  should  be  non-absorbent  and 
of  the  best  quality,  i.  e.,  as  free  from  foreign  matter  as  possible. 
The  plugs  should  be  sufficiently  loose  to  permit  the  interchange  of 
gases  and  at  the  same  time  tight  enough  to  support  the  weight  of 
the  vessel  and  its  contents,  otherwise  they  are  apt  to  be  pulled  out 
in  handling  the  vessels.  The  cotton  should  be  rolled  into  a  cylin- 
der of  the  proper  diameter  and  long  enough  to  extend  into  the 
mouth  about  2y2  cm.  (1  in.)  and  project  sufficiently  to  protect  the 
lips  from  dust.  The  plug  should  be  pushed  in  straight  and  not 
twisted;  the  surface  next  to  the  glass  must  be  perfectly  smooth, 
presenting  no  creases  for  the  entrance  of  dust. 

REFERENCES.     A.  127;  H.  44;  M.  &  R.  49;  McF.  164;  P.  223. 

SPECIAL  DIRECTIONS.  Plug  all  test-tubes,  flasks  and  fermenta- 
tion tubes  in  your  possession. 

EXERCISE  3.     STERILIZATION  OF  GLASSWARE. 

GENERAL  DIRECTIONS.  The  glassware  thus  prepared  is  ready 
for  sterilization,  which  process  is  accomplished  in  an  apparatus 
called  the  hot  air  sterilizer.  This  is  a  sheet  iron  or  copper  box  with 

a  double  wall  which  permits  of  rapid 
heating.  The  apparatus  should  be  so  ar- 
ranged that  a  temperature  of  150°  C.  can 
be  quickly  reached  and  readily  main- 
tained. In  such  a  sterilizer  all  glassware 
L  to  be  used  for  the  reception  of  culture 
media,  such  as  flasks,  test-tubes,  Petri 
dishes,  etc.,  is  submitted  to  a  temperature 
of  140-150°  C.  for  1  hour,  or  until  the 
cotton  plugs  are  slightly  browned;  this 
change  being  due  to  the  incipient  char- 
ring of  the  cotton.  The  test-tubes  are 
placed  erect  in  square  baskets  made  of 
galvanized  iron  wire.  When  the  air  in 
the  sterilizer  has  cooled  to  about  40°  C. 
the  glassware  can  be  taken  out  and  stored  ready  for  use.  The  Petri 
dishes  are  not  to  be  opened  until  used  for  culture  purposes. 

REFERENCES.  A.  75  and  127 ;  H.  36 ;  M.  &  R.  29 ;  McF.  164 ; 
P.  223. 

;x 


Fig.  1.    Hot  Air  Sterilizer, 
(Muir  &  Ritchie). 


6 


GENERAL  BACTERIOLOGY 


SPECIAL  DIRECTIONS.  All  glassware  prepared  in  1.  is  to  be 
sterilized  for  one  hour  at  150°  C.  The  small  pipettes  should  be 
placed  in  brass  tubes,  provided  for  the  purpose,  and  also  sterilized. 


EXERCISE    4.     PREPARATION   OF   BOUILLON. 

GENERAL  DIRECTIONS.  Any  one  of  the  three  methods  (A,  B 
or  C)  may  be  used.  They  are  arranged  in  order  of  preference,  but 
method  C  is  the  most  convenient,  and  hence  most  used. 


A. 

a.  From  500  grams  (1%  Ibs.)  of  lean 
beef  remove  the  fat  and  connective  tis- 
sue and  mince  (or  use  Hamburg  steak). 


b.  Add  1  liter  of  distilled  water  and 
after    shaking    thoroughly    set    in    ice 
chest  for  12  to  24  hours. 

c.  Squeeze   through   a   cloth   and   add 
enough    distilled    water    to    filtrate    to 
make  1  liter  and  place  in  vessel  to  cook. 


C. 

a.  Weigh  out 
three  grams  of 
beef  extract 
(such  as  Lie- 
big's). 

6.  Add  1  liter 
of  distilled  wa- 
ter. 

c.  Place  in 
vessel  for  cook- 
ing. 


B. 
a.  Ditto. 


b.  Add  1  liter  of 
distilled   water. 

c.  Place   in   ves- 
sel    for     cooking, 
then    cook    for    ys 
hour  at  about  70° 
C.,    filter    through 
paper     and     make 
up  to  1  liter. 


d.  Add  to  any  of  the  above  solutions:  \%   (10  gms.)  peptone 
(Witte)  and  %%  (5  gms.)  common  salt  (NaCl),  then  weigh  solu- 
tion, with  vessel,  so  that  the  water  which  is  subsequently  driven 
off  in  cooking  can  be  accurately  replaced. 

Cooking  may  be  done  either  in  a  flask  which  is  heated  in  a  water- 
bath  or  sterilizer,  double-walled  boiler,  or  rice-cooker.  In  case  a  rice- 
cooker  is  used  a  50%  solution  of  calcium  chloride  should  be  placed 
in  the  outer  vessel  instead  of  water  as  by  this  means  the  contents 
of  the  inner  vessel  can  be  brought  to  a  rapid  ebullition,  something 
impossible  by  the  use  of  water  alone. 

e.  Heat,  not  above  60°  C.,  until  ingredients  are  in  solution, 
then  restore  the  water  lost  by  evaporation. 

/.  Neutralize.  This  is  a  very  important  step  and  calls  for  great 
care.  Of  the  following  methods,  A  is  more  accurate  and  should 
be  employed  for  special  or  research  work.  For  ordinary  routine 
work  B  may  be  employed. 


GENERAL  BACTERIOLOGY 


A. 

1.)  Titrate  as  follows:  Pipette  off  5 
cc.  of  the  fluid  into  a  10  cm.  evaporating 
dish,  add  45  cc.  of  distilled  water,  boil 
for  three  minutes,  add  1  cc.  of  phen- 
olphthalein (0.5%  substance  in  50%  al- 
cohol), and  then  run  in  carefully,  drop 
by  drop,  from  a  burette  a  twentieth 
normal1  solution  of  sodium  hydroxide 
(^NaOH)  until  the  solution  turns  a 
faint  pink  color.  Treat  two  other  sam- 
ples in  the  same  way.  If  the  amount  of 
NaOH  required  is  approximately  the 
same  in  each  case  the  average  can  be 
taken  as  the  amount  necessary  to  neu- 
tralize 5  cc.  Calculate  the  amount  nec- 
essary to  neutralize  the  whole  (1000—15 
cc.).  Since  this  amount  would  dilute 
the  medium  too  much,  a  stronger  solu- 
tion (normal)  is  used,  hence, 


Use  a  normal1  solution  of  so- 
dium hydroxide  (£NaOH).  Add 
to  the  hot  solution  a  few  cc.  at  a 
time  at  first,  later  a  few  drops, 
stirring  thoroughly  with  a  glass 
rod.  After  each  addition,  test 
by  placing  a  drop  of  the  solution 
by  means  of  the  glass  rod  on  a 
strip  of  phenolphthalein  paper. 
(Prepared  by  dipping  filter  pa- 
per in  a  solution.)  The  addition 
should  continue  until  the  test  pa- 
per is  turned  a  faint  pink  color. 


2.)  Neutralize  by  adding  -gVtli  of  the  volume  calculated  above 
of  a  normal  solution  of  sodium  hydroxide.  Test  the  accuracy  of 
the  work  at  this  point  by  the  addition  of  a  few  drops  of  phenol- 
phthalein to  a  cc.  or  so  of  the  medium.  If  a  faint  pinkish  tint  is 
not  obtained,  titration  and  neutralization  must  be  repeated. 

g.  Boil  for  5  minutes  and  restore  weight. 

h.  Test  reaction  and  adjust  if  necessary. 

i.  Add  0.5  to  1.5%  of  a  normal  hydrochloric  acid.  The  amount 
of  acid  to  be  added  varies  with  the  purpose  for  which  the  medium 
is  to  be  used,  e.  g.,  in  water  analysis  +  1.0  (acid)  is  preferable, 
with  the  pathogenic  bacteria  a  smaller  amount  of  acid  (J-0.5) 
more  nearly  meets  requirements. 

j.  Heat  until  precipitate  appears  flaky  and  then  filter  through 
moistened  filter  paper.  (For  method  of  folding  see  Abbott  p.  96). 

The  filtrate  (bouillon)  should  be  of  a  light  straw  color,  per- 
fectly clear,  and  should  not  give  a  precipitate  on  boiling. 

REFERENCES.  A.  94;  H.  45;  M.  &  B.  35;  McF.  180;  P.  212; 
P.  B.  C.  18-24. 

SPECIAL  DIRECTIONS.  Prepare  1  liter  of  bouillon  according  to 
method  C.  Secure  and  put  to  soak  meat  for  7.  See  Rule  IV. 


1  Normal  solutions  are  prepared  so  that  one  liter  at  16°  C.  shall  contain 
the  hydrogen  equivalent  of  the  active  reagent  weighed  in  grams  (Sutton). 
For  present  purposes  a  4%  solution  of  sodium  hydrate  is  sufficiently  accurate. 


10 


GENERAL  BACTERIOLOGY 


EXERCISE  5. 


FILLING  TEST-TUBES  AND  FLASKS  WITH  CULTURE 
MEDIA. 


GENERAL  DIRECTIONS.  In  filling  tubes  be  careful  not  to  allow 
the  media  to  touch  the  neck  of  the  vessel  as  this  will  cause  the 
cotton  to  stick  to  the  glass  when  the  plugs  are  removed.  Place 
the  culture  fluid  to  be  tubed  in  a  funnel  arranged  with  a  delivery 
tube  and  stopcock  (Fig.  2),  from  which  it  can  be 

run  into  sterile  vessels.     Test-tubes  should  contain      _ _ 

6-10  cc.  of  medium   (about  3  cm.  deep).     Flasks 
are  to  be  filled  about  three-fourths  full. 

SPECIAL    DIRECTIONS.     Fill    15    test-tubes    and 
preserve  remainder  of  bouillon  in  flasks. 


EXERCISE  6.     STERILIZATION  OF  CULTURE 
MEDIA. 

EXPLANATORY.  To  sterilize  culture  media  steam 
is   used   almost   exclusively   either   as   streaming        FIG.  2.  Appara- 
steam  or  under  pressure.     The  unconfined  steam     tubes* 
is  applied  in  an  apparatus  known  as  a  steam  ster- 
ilizer.    Of  the  various  patterns  the  Arnold  is  perhaps  the  most 
satisfactory.     It  is  effective,  economical  in  the  use  of  gas,  and 
does  not  allow  the  escape  of  large  quantities  of  steam  into  the 
room  as  a  large  part  is  condensed  to  be  reconverted  into  steam. 

A  simple  steam  sterilizer  is  shown  in 
>••      Fig.  3,  and  for  student  use  is  very 
convenient.     The    method    of    using 
either    form    is    identical.     Always 
have  plenty  of  water  present  before 
heating.     Exposure  is  made  on  three 
consecutive  days  for  20  minutes,  be- 
ginning to  count  time  when  the  ma- 
o«    terial  reaches  the  temperature  of  the 

FIG.  3.    Simple  sterilizer  consisting    steam>  which  wil1  varv  with  different 

of  a  galvanized  iron  pail  with  a  cover  substances  and  the  volume  treated. 

a  ana  a  raise  bottom  o. 

Between  successive  steamings  culture 

media  should  be  kept  under  conditions  favorable  to  spore  germi- 
nation,' i.  e. ,  at  the  room  temperature.  This  method  of  steriliza- 
tion is  known  as  the  discontinuous  method  or  Tyndalization. 

For 'the  employment  of  steam  under  pressure  the  autoclave  is 
essential.     The  lid  should  contain  a  thermometer  as  well  as  a 


12 


GENERAL  BACTERIOLOGY 


steam  gauge,  safety  and  outlet  valve.  A  thermo-regulator  is  also 
desirable.  The  following  table  gives  the  temperature  correspond- 
ing to  atmospheres  of  pressure  indicated  on  the  gauge : 


TABLE  OF  TEMPERATURES  CORRESPONDING  TO  STEAM- 
PRESSURES. 


Temperatures 

Steam-Pressure 

Temperatures 

Steam-Pressure 

F. 

C. 

Lbs. 

F. 

C. 

Lbs. 

212° 

100° 

0 

251° 

121.5° 

15 

228° 

109° 

5 

260° 

126.5° 

20 

240° 

115.5° 

10 

287° 

141.5° 

40 

This  table  is  true  only  when  all  of  the  air  in  the 
apparatus  is  replaced  by  steam,  and  hence  the  steam 
must  be  allowed  to  escape  freely  before  the  outlet 
valve  is  closed.  A  single  exposure  of  20  minutes  at 
a  temperature  of  120°  C.  (one  additional  atmos- 
phere) is  sufficient  to  kill  all  germ  life.  After  the 
proper  exposure,  care  must  be  taken  not  to  allow  the 
steam  to  escape  too  rapidly,  otherwise  the  culture 
media  may  be  forced  against  the  plugs  owing  to  the 
unequal  pressure. 

GENERAL  DIRECTIONS.  Ordinary  media  may  be 
sterilized  by  either  method.  In  case  of  gelatin  and 
sugar  media  the  temperature  should  not  exceed 
110°  C.  for  15  minutes. 


O    O    O  0  < 


REFERENCES. 
166;  P.  218. 


FIG.  4.  Autoclave; 

A.  59-77  ;  H.  37  ;  M.  &  E.  29  ;  McF.  a,    safety  valve;    &, 

blow-off  pipe;  c. 
gauge;  (M  u  i  r  & 
Kitchie). 


SPECIAL    DIRECTIONS.     Sterilize    bouillon    pre- 
pared in  4  for  20  minutes  in  a  steam  sterilizer  on  three  consecutive 
days,  or  in  the  autoclave  at  120°  C.  for  20  minutes.     Rule  V. 

N.  B.  Some  time  is  required  to  raise  the  temperature  of  the 
media  to  that  of  the  steam,  especially  if  the  vessels  are  large. 

All  media  should  be  carefully  examined  every  day  for  a  week  or 
more,  and  if  "specks"  or  the  least  cloudiness  appears,  the  medium  is 
not  sterile  and  the  process  of  sterilization  must  be  repeated. 


14  GENERAL  BACTERIOLOGY    • 

EXERCISE  7.     PREPARATION  OF  GELATIN. 

GENERAL  DIRECTIONS. 

Same  as  bouillon  (4). 
c. 

d.  Add  \%  peptpne,  0.5%  salt  and  10-15 %*  of  the  best  gold 
label,  sheet  gelatin,  and  weigh. 

e.  Heat  until  ingredients  are  dissolved. 
/.  Neutralize. 

g.  Boil  5  minutes  and  restore  weight. 

h.  Test  reaction. 

i.  Boil  until  albumin  coagulates  and  floats  in  the  clear  fluid.  If 
beef  extract  is  used  it  will  be  necessary  to  first  cool  below  60°  C. 
and  thoroughly  stir  in  an  egg. 

j.  Filter.  Arrange  the  apparatus 
shown  in  Fig.  5.  Use  absorbent  cotton. 
The  funnel  and  flask  should  first  be  heated 
with  warm  water.  Usually  the  hot  gelatin 
will  filter  without  the  use  of  the  pump.  If 
the  pump  is  needed  it  should  be  started  be- 
fore pouring  in  the  culture  medium.  This 
prevents  the  unfiltered  gelatin  from  pass- 
ing between  the  cotton  and  glass. 

Jc.  Add  5.0  cc.  (0.5  %)  of  a  normal  hy- 

,        1  -I      •          •  i       i    ,•  FIG.  5.    Apparatus  for  fllter- 

arOCnloriC  acid.  SOlUtlOn.  ing  media  through  absorbent 

7     m    v       /K\  cotton;    a,  coil   of  wire  over 

I.    lUDe.   (D).  laid  with  layer  of   absorbent 

0,      .,.           .         ^  „           OA  cotton;    b,  tubes  for  making 

m.    Sterilize      in      the  Steamer      for      20   connection  with  air  pump;   c, 

minutes  on  three  consecutive  days  or  in  the  ^ance^/waterlnto^Isks.  er 
autoclave  at  110°  C.  for  15  minutes. 

REFERENCES.  A.  99 ;  H.  47 ;  M.  &  R.  40 ;  McF.  184 ;  P.  215 ; 
P.  B.  C.  26. 

SPECIAL  DIRECTIONS.  Make  1  liter,  using  method  A.  Fill  30 
test-tubes.  Put  the  remainder  in  flasks,  sterilize  in  steam  sterilizer 
or  autoclave.  Remember  long  exposure  to  high  heat  injures  the 
solidifying  properties  of  gelatin. 


1  The  amount  to  be  varied  according  to  the  season  of  the  year,  10  per  cent, 
in  winter,  12-15  per  cent,  in  summer,  but  it  should  be  remembered  that  differ- 
ent quantities  affect  the  appearance  of  the  cultures. 


16  GENERAL  BACTERIOLOGY 

EXERCISE  8.  PREPARATION  OF  AGAR  (RAVENED). 

GENERAL  DIRECTIONS. 

Add  15  grams  of  agar-agar  threads  (finely  chopped;  to  500  cc. 
of  water  and  either  (1)  dissolve  in  autoclave  by  heating  up  to  120° 
C.,  closing  off  gas  and  allowing  to  cool,  or  (2)  boil  until  the  agar-agar 
is  dissolved  (about  y2  hour)  and  make  up  loss  of  water  by  evapora- 
tion. While  the  agar  is  being  dissolved  proceed  as  follows : 

a.  Same  as  in  the  preparation  of  bouillon  (4  a). 
fc.  Add  500  cc.  of  distilled  water. 

c.  Same  as  bouillon  (4  c). 

d.  Add  10  gms.  of  peptone  and  5  gins,  of  salt. 

e.  Heat  until  peptone  is  dissolved. 
/.  Neutralize. 

g.  Cool  to  60°  C.,  add  agar  solution  and  mix  (in  case  extract  is 
used  it  will  be  necessary  to  add  an  egg  at  this  point) . 

h.  Boil  until  albumin  is  coagulated  and  floats  in  the  clear  liquid 
and  restore  weight. 

i.  Test  reaction. 

,;'.  Add  0.5%  normal  hydrochloric  acid. 
~k.  Filter  as  in  case  of  gelatin.   (7j.) 
I  Tube. 

m.  Sterilize  in  steam  for  15  minutes  on  three  successive  days  or 
in  autoclave  for  20  minutes  at  120°  G. 
After  the  last  sterilization  place  most  of 
the  tubes  in  a  sloping  position  to  harden 
(Fig.  6) ,  these  are  known  as  agar  slopes. 
Those  solidified  in  an  upright  position,   — 
frequently  called  "deep   stick  agar,"     »»•«•  Method  of  sloping  agrar, 
are  used  to  make  plate  cultures. 

REFERENCES.  A.  104 ;  H.  47 ;  M.  &  R.  38 ;  McF.  185 ;  P.  215 ; 
P.  B.  C.  27 ;  Journal  of  Applied  Microscopy,  1898,  1 ;  106. 

SPECIAL  DIRECTIONS.  Use  meat  extract,  make  1  liter,  fill  25 
tubes  and  after  last  sterilization  incline  20  of  them.  Place  the 
remainder  in  flasks  and  sterilize. 


18  GENERAL  BACTERIOLOGY 

EXERCISE  9.     PREPARATION  OF  POTATOES  (BOLTON). 

GENERAL  DIRECTIONS. 

a.  Select  a  number  of  rather  large  test-tubes    (150x20  mm.), 
place  a  small  wad  of  absorbent  cotton1  in  the  bottom 
of  each  (Fig.  la),  plug  and  sterilize  as  usual. 

~b.  Wash  a  large  potato,  then  with  a  cork  borer 
slightly  smaller  than  the  test-tubes  punch  out  cylinders 
about  5-6  cm.  long. 

c.  Divide  these  diagonally  and  trim  to  shape  indi- 
cated in  Fig.  7  &. 

d.  Add  a  few  drops  of  distilled  water  to  each  test- 
tube  and  place  pieces  of  potato  in  position. 

e.  Sterilize  on  three  consecutive  days  for  30  to  45 

minutes.  FlG.7.  Boiton's 

Unless  the  tubes  are  to  be  used  immediately,  they      potato  tube' 
should  be  sealed.     (11.)   The  dark  color  can  be  prevented  by  im- 
mersing the  pieces  between  c  and  d  in  running  water  for  12-18  hours. 

REFERENCES.  A.  107 ;  H.  49  ;  M.  &  .  47 ;  McF.  190 ;  P.  216 ;  P. 
B.  C.  28 ;  S.  47. 

SPECIAL  DIRECTIONS.  Prepare  15  test-tubes  of  potato,  sterilize 
and  seal  with  paraffin.  (11.2.) 

EXERCISE  10.     PREPARATION  OF  WATER-BLANKS. 

GENERAL  DIRECTIONS.  Water-blanks  are  prepared  by  placing 
exactly  10  cc.  of  a  physiological  salt  solution  (6  gms.  per  1,000  cc. 
water)  in  test-tubes  and  sterilizing  in  autoclave  15  minutes  at  120° 
C.,  or  in  steamer  15  minutes  on  three  successive  days. 

SPECIAL  DIRECTIONS.     Prepare  and  sterilize  10  water-blanks. 

EXERCISE  11.     CARE  OF  CULTURE  MEDIA. 

When  sterile  culture  media  (or  test-tube  cultures)  are  to  be 
kept  for  some  time  they  must  be  protected  from  evaporation  and 
stored  in  a  dark,  cool  place.  Evaporation  may  be  checked  to  a  con- 
siderable extent,  (1)  by  storing  them  in  tin  cans,  e.  g.  quinine  cans. 
Care  must  be  taken,  however,  that  these  do  not  become  too  damp  in 
which  case  the  mould  fungi  frequently  grow  through  the  cotton 
plugs;  (2)  flasks  and  test-tubes  may  be  sealed  by  removing  the 


1  Gage  recommends  glass  beads.     A  smaller  cylinder  of  potato  may  also  be 
used  instead  of  the  cotton;  in  this  case  the  tubes  would  be  sterilized  empty. 


20  GENERAL  BACTERIOLOGY 

plugs,  dipping  same  in  melted  paraffin  (melting  point  about  50°  C.) 
and  then  replacing  them;  (3)  by  cutting  off  the  projecting  cotton 
and  drawing  over  the  mouth  of  the  vessel  a  rubber  cap  (made  for  the 
purpose)  which  has  been  sterilized  in  a  solution  of  mercuric  bi- 
chloride, or  rubber  dam,  easily  obtained  from  dentists,  fastened  on 
with  a  rubber  band,  may  also  be  used ;  or  (4)  by  use  of  a  cap  of  tin- 
foil. In  this  case  the  foil  should  be  put  on  as  soon  as  the  tubes  are 
filled,  and  sterilized  with  the  medium. 

All  media  should  be  carefully  examined  every  day  for  a  week  or 
more,  and  if  spots  or  the  least  cloudiness  appears,  the  medium  is  not 
sterile  and  the  process  of  sterilization  must  be  repeated. 

All  receptacles  containing  media  should  be  labeled  after  steriliza- 
tion. For  this  purpose  labels  can  be  purchased,  the  size  used  for 
glass  slides,  or  gummed  paper  in  sheets  can  be  cut  into  squares 
( 2  cm. ) .  The  labels  are  to  be  attached  to  each  ves- 
sel 1  cm.  from  the  lip.  The  name  of  the  student, 
the  kind  of  medium  and  the  date  of  preparation 
should  be  written  across  the  top,  leaving  the  rest  of 
the  label  to  be  filled  in  when  the  medium  is  inocu- 
lated. Rule  VII. 

EXERCISE  12.     PLATINUM  NEEDLES. 

GENERAL  DIRECTIONS.     These  are  made  by  fusing  a  piece  of  No. 
27  platinum  wire   (5  cm.  long)   into  a  glass  rod  or  tube   (18  cm. 
f  ^      long).      (Fig.  8.)     The  dan- 
ger of  having  the  wire  crack 

O- c  ]      off  when  the  needle  is  heated 

FIG.  8.   Platinum  Needles.  is  lessened  if  a  little  piece 

(1/2  cm.)  of  fusible  glass  is  soldered  on  the  glass  rod  before  the 
wire  is  melted  in.  Each  student  should  have  two  such  needles ;  in 
one  the  wire  should  be  straight  (designated  " needle")  and  the  other 
bent  to  form  a  ' '  loop. ' '  This  loop  should  be  formed  around  a  No.  10 
wire.  These  instruments  must  be  sterilized  shortly  before  and  im- 
mediately after  use  by  heating  the  wire  to  a  glow  in  the  gas  flame. 
The  handle  should  also  be  passed  through  the  flame  two  or  three 
times.  Cool  before  using.  If  the  habit  of  sterilizing  is  thoroughly 
acquired  much  trouble  will  be  avoided  and  possible  danger  prevented. 
These  needles  will  be  in  constant  use. 

REFERENCES.  A.  131 ;  H.  42 ;  M.  &  R.  51 ;  McF.  196 ;  P.  B.  C.  33, 
foot  note. 


22 


GENERAL  BACTERIOLOGY 


EXERCISE  13.     TEST-TUBE  CULTUEES. 

EXPLANATORY.  The  extreme  minuteness  and  slight  variation  in 
the  form  of  different  bacteria  render  a  thorough  study  of  them  by 
direct  microscopic  observation  a  difficult  and  well  nigh  impossible 
task.  In  their  study,  therefore,  it  is  necessary  to  depart  from  the 
usually  accepted  rules  that  govern  the  determination  of  the  life 
history  of  other  forms  of  life  and  resort  to  special  methods.  The 
most  successful  of  these  are  those  known  as  culture  methods.  Ac- 
cording to  these  methods  the  bacteria  are  sown  on  various  food  sub- 
stances and  upon  these  they  develop  forming  masses  easily  visible 
to  the  naked  eye.  The  manner  of  their  growth  and  the  changes 
which  they  produce  in  these  media  make  it  possible  to  detect  differ- 
ences which  would  otherwise  escape  attention.  The  most  common 
culture  media,  bouillon,  gelatin,  agar,  and  potato  have  already  been 
prepared,  and  others  will  be  described  as  needed. 

Cultures  may  be  made  either  in  test-tubes  (streak  or  stab  cult- 
ures), or  on  glass  plates,  as  plate  cultures.  The  plate  culture  is 
especially  important  and  is  used  (a)  to  obtain  pure  cultures;  and 
(6)  for  ascertaining  the  character  of  the  colonies  as  an  aid  to 
their  diagnosis.  The  tube-cultures  are  serviceable  in  giving  oppor- 
tunity for  a  further  study  of  the  characters  as  well  as  to  furnish  the 
most  convenient  method  of  maintaining  the  cultures. 

GENERAL  DIRECTIONS.  Bacteria  when  obtained  in  "pure 
culture"  are  usually  grown  in  test-tube  cultures.  To  make  these 
a  small  portion  of  a  previous  culture  is  transferred  to  fresh  culture 
media  by  means  of  the  platinum  needles. 

a.  Stab  Cultures  are  made  in  test-tubes  containing  solid,  trans- 
parent media,  such  as  gelatin 
and  agar.  The  end  of  a  ster- 
ile needle  is  infected  with  the 
material  to  be  transferred. 
The  needle  is  then  thrust  into 
the  medium  to  the  bottom  of 
the  test-tube  and  withdrawn. 
In  this  way  the  bacteria  are 
left  along  the  entire  length 
of  the  needle  track.  For 
method  of  holding  tubes  see 
Fig.  9.  They  are  held  in  an 
inclined  position  to  prevent 

the    possibility    of    infection         ^  g    Method  of  holding  test.tubes. 
from  the  air.  «,  cotton  plugs. 


24  GENERAL  BACTERIOLOGY 

b.  Streak  Cultures  are  cultures  made  by  drawing  the  needle,  or 
better,  the  loop,  over  the  surface  of  the  medium  (test-tubes  with 
media  having  sloped  surfaces  or  plate  cultures).     Agar,  potato  and 
blood  serum  are  frequently  used  in  this  way,   and  occasionally 
gelatin. 

c.  Liquid  Cultures  (bouillon,  milk,  etc.)  are  inoculated  by  trans- 
ferring the  desired  material  to  them  on  either  the  needle  or  loop. 

REFERENCES.     A.  152 ;  H.  58 ;  M.  &  R.  51 ;  McF.  198. 

SPECIAL  DIRECTIONS. 

a.  Make  a  gelatin  stab,  an  agar  streak,  a  potato  streak,  and  a 
bouillon  culture  of  Bacil lus  subtilis  (EHRENB.)  COHN  (hay  bacillus) 
and  Bacillus  coli  (Escn.)  MIG.  (colon  bacillus)  from  agar  cultures 
supplied.     Rule  VIII. 

b.  Label  each  tube,  writing  the  name  of  the  organism,  the  date  of 
inoculation  and  your  own  name.     Rule  VII. 

c.  Place  the  gelatin  in  the  cool  chamber,  and  the  other  cultures  in 
the  incubator  at  28°  C.     See  next  Exercise. 

EXERCISE  14.  INCUBATION  OF  CULTURES. 

EXPLANATORY.  Most  bacteria  grow  at  ordinary  temperatures 
(22°  C.),  but  their  growth  is  usually  hastened  by  a  higher  tempera- 
ture (e.  g.  28°-30°  C.)  The  pathogenic,  or  disease-producing  bac- 
teria grow  best  at  the  temperature  of  the  human  body  (38°  C.). 
All  bacteriological  laboratories  are,  therefore,  supplied  with  appara- 
tus arranged  for  maintaining  constant  temperatures,  known  as 
thermostats  or  incubators. 

The  non-pathogenic  cultures  are  usually  kept  at  28°  C.,  while 
the  pathogenic  ones  are  kept  at  38°  C.  All  gelatin  cultures,  how- 
ever, must  be  kept  at  a  temperature  several  degrees  below  the  melt- 
ing point  of  gelatin,  i.  e.,  not  above  22°  C.  Ordinarily  the  tempera- 
ture of  the  locker,  especially  near  the  floor,  will  be  found  satisfac- 
tory. In  a  very  warm  room,  particularly  in  the  summer,  an  artifi- 
cially cooled  chamber  will  be  necessary. 

Test-tube  cultures  are  stored  in  the  various  incubators  in  tin 
cans  or  glass  tumblers  with  a  layer  of  cotton  in  the  bottom,  while 
the  Petri  dishes  are  stacked  in  low  piles. 

REFERENCES.     A.  Ch.  VIII ;  H.  55  ;  M.  &  R.  82 ;  P.  231. 

SPECIAL  DIRECTIONS. 

a.  Incubate  all  cultures  of  the  non-pathogenic  bacteria  at  28°  C., 


BACILLUS  STJBTILIS  (EHRENB.)  COHN  25 


Gelatin  Stab:    Grown  24hours  at °C. 


48  hours  at 


=c. 


6  days  at <>C. 


Agar  Streak:     Grown  24  hours  at °C. 


I 


!   I 


48  hours  at 


6  days  at °C. 


Potato:    Grown  24  hours  at 


9 

•s 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at. 


26  GENERAL  BACTERIOLOGY 

except  the  gelatin.  Keep  these  in  the  cool  chamber.  After  growth 
has  taken  place,  the  cultures  can  be  taken  from  the  incubator  and 
kept  at  the  room  temperature. 

1}.  Study  and  make  diagrams  of  an  incubator,  a  Reichert  thcrmo- 
regulator,  a  Roux  thermo-regulator  and  Koch 's  safety  burner. 

EXERCISE  15.     STUDY  OF  TEST-TUBE  CULTURES. 

GENERAL  DIRECTIONS.  As  soon  as  growth  becomes  visible  a  sys- 
tematic and  careful  study  of  the  cultures  should  be  made.  A  de- 
tailed list  of  the  points  to  be  noted  will  be  found  in  Chapter  III, 
and  should  be  consulted  in  writing  up  the  descriptions.  The  sum- 
mary below  will,  however,  be  found  useful. 

For  bouillon  cultures  note :  1 )  condition  of  fluid,  2 )  character 
of  sediment,  3)  presence  or  absence  of  membrane,  and  4)  charac- 
teristic odor. 

For  solid  cultures  (agar  and  potato  slopes),  note:  1)  Form  of 
growth,  2)  size,  3)  surface  elevation,  4)  consistency,  5)  color,  6) 
effect  on  media,  and  7)  characteristic  odor. 

For  gelatin  stab  cultures,  note :  1)  Effect  on  media,  a.  non-lique- 
fying, i)  line  of  puncture,  ii)  surface,  &.  liquefying,  i)  shape  of 
liquefied  area,  ii)  condition  of  fluid,  iii)  character  of  sediment,  2) 
characteristic  odor. 

The  study  should  be  continued  from  day  to  day  as  long  as 
changes  are  noted.  Make  drawings  wherever  they  will  be  of 
service  in  elucidating  the  descriptions. 

SPECIAL  DIRECTIONS.  Study,  write  careful  descriptions  and 
make  necessary  drawings  of  all  the  cultures  made.  For  recording 
results  use  the  table  on  pages  25  and  27. 


EXERCISE  16.     CLEANING  SLIDES  AND  COVER  GLASSES. 

GENERAL  DIRECTIONS.  Slides  can  be  sufficiently  cleaned  by 
washing  in  water  or  alcohol  and  drying  with  a  towel.  The  cover- 
glasses  for  bacteriological  work,  however,  must  not  only  be  freed 
from  visible  dirt  but  must  be  rendered  free  from  fat.  One  of  the 
best  methods  is  the  following:  New  cover-glasses  are  cleaned  by 
washing  in  water  and  drying  from  alcohol  by  rubbing  them  between 
driers  (two  wooden  blocks  20x10x2^  mm.  covered  with  several 
layers  of  cotton  cloth  or  chamois  skin),  and  then  heating  them  on 


BACILLUS  COLI  (Escn.)  MIG. 


Gelatin  Stab:    Grown  24  hours  at. 


5C. 


Agar  Streak:    Grown  24  hours  at 


O  i 


48  hours  at 


6  days  at                                       oC 

6  days  at °C. 


Potato:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Bouillon:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at.. 


28  GENERAL  BACTERIOLOGY 

a  piece  of  sheet  iron  or  in  hot  air  sterilizer  for  one  hour  at  about 
200°  C.  They  are  best  kept  in  a  clean  Petri  dish  and  handled 
with  forceps.  (Novy).  Old  slides  and  covers  having  balsam  on 
them  should  first  be  dropped  one  by  one  into  a  cleaning  solution 
(potassium  bichromate  60,  sulphuric  acid  60,  water  1,000),  and 
boiled  for  one-half  hour  and  then  treated  as  above. 

SPECIAL  DIRECTIONS.     Clean  y2  oz.  of  cover-glasses  and  place 
them  in  a  clean  Petri  dish. 


EXERCISE  17.     PREPARATION  OF  STAINING  SOLUTIONS. 

GENERAL  DIRECTIONS.  The  dyes  most  useful  for  staining  bac- 
teria are  the  basic  anilin  dyes  which  come  in  powdered  or  crystal- 
line form.  (Gruebler's  dyes  are  standard).  Those  in  most  com- 
mon use  are  Fuchsin,  Methylen  blue,  Gentian  violet  and  Bismarck 
brown.  They  keep  in  powdered  form,  with  perhaps  the  exception 
of  Methylen  blue,  but  because  of  greater  convenience  and  equally 
good  keeping  qualities,  saturated  alcoholic  solutions  are  kept  in 
stock.  These  are  made  by  adding  the  dry  dye  to  95%  alcohol  to 
saturation  and  filtering.  This  form  can  not  be  used  for  staining 
bacteria.  The  following  solutions  are  required  to  begin  work  with : 

1.  Aqueous  solution  of  Gentian  violet. 

Saturated  alcoholic  solution  of  Gentian  violet 2.5  cc. 

Distilled  water 47.5  cc. 

2.  Saturated  aqueous  solution  of  Bismarck  brown. 

3.  Ziehl's  carbol-fuchsin. 

Saturated  alcoholic  solution  of  Fuchsin 5  cc. 

Solution  of  carbolic  acid  (5%) 45  cc. 

4.  Loeffler's  Methylen  blue. 

Saturated  alcoholic  solution  of  Methylen  blue 15  cc. 

Potassium  hydrate  (IrlOjOOO)1 50  cc. 

5.  Ehrlich's  Anilin  Oil  Gentian  violet.2 

Saturated  alcoholic  solution  of  Gentian  violet 6  cc. 

Absolute  alcohol 5  cc. 

Anilin  water  .  50  cc. 


1  This  dilution  can  be  readily  made  by  taking  1  cc.  of  a  10%  potassium 
hydrate  solution,  making  this  up  to  100  cc.,  then  taking  5  cc.  of  this  and 
making  it  up  to  50  cc. 

2  Some  prefer  anilin  oil  made  as  follows :   Solution  A.,  2  cc.  of  anilin  oil 
and  100  cc.  of  distilled  water;  Solution  B.,  25  cc.  of  filtered  saturated  alco- 
holic  solution  of   gentian  violet  ,and   75   cc.  of  Solution  A.     Mix  and  filter. 
This  stain  remains  good  for  a  long  time. 


30  GENERAL  BACTERIOLOGY 

Anilin  water  is  prepared  by  adding  2-3  cc.  of  anilin  oil,  drop  by  drop,  to 
50  ec.  of  water,  thoroughly  shaking  and  then  filtering  through  moistened 
paper  until  perfectly  clear. 

This  stain  should  stand  24  hours  and  then  be  filtered.  It  does  not  keep 
well  and  must  not  be  used  when  more  than  14  days  old. 

6.  Gram's  Iodine  solution. 

Iodine  1  gm. 

Potassium  iodide   2  gm. 

Distilled  water 300  cc. 

7.  Gabbett's  Methylen  blue  solution. 

Methylen  blue   (dry) 2  gms. 

Sulphuric  acid 25  cc. 

Distilled  water 75  cc. 

8.  Alcohol,  96%. 

REFERENCES.     A.  163  ;  H.  85  ;  M.  &  R.  97 ;  P.  200. 

SPECIAL  DIRECTIONS.  Prepare  the 
solutions  of  dyes  from  the  saturated 
alcoholic  solutions  (furnished)  and 
place  them  in  2  oz.  bottles  arranged 
with  pipettes  and  neatly  labeled.  The 
bottles  are  conveniently  kept  in  a 
block.  Fig.  10. 


EXERCISE  18.     SIMPLE  COVER-GLASS 

PREPARATION  FIG.  10.    Block  for  stain  bottles. 

GENERAL  DIRECTIONS.  Bacteria  may  be  studied  under  the  micro- 
scope in  a  living  condition  in  a  hanging  drop  preparation  (21)  ; 
but  on  account  of  their  hyaline  character,  which  makes  the  exami- 
nation difficult,  the  student  should  first  learn  to  stain  them  and 
later  make  the  hanging  drop  preparation.  With  a  few  exceptions 
all  bacteria  can  be  stained  by  the  following  process :  A  small  drop 
(about  the  size  of  a  pinhead)  of  distilled  water  is  placed  on  a  clean 
cover-glass  by  means  of  the  platinum  loop.  With  a  sterile  needle  a 
portion  of  the  material  to  be  examined  is  secured  and  while  the 
cover-glass  is  held  in  the  fingers  of  the  left  hand  the  bacteria  on  the 
needle  are  introduced  into  the  water,  thoroughly  mixed  and  then 
spread  in  a  thin  film  over  as  much  of  the  surface  of  the  cover-glass 
as  possible.  When  the  bacteria  are  taken  from  fluid  media  a  drop 
of  water  will  not  be  necessary.  In  this  case  use  a  loop.  The  film 
is  now  allowed  to  dry.  If  the  drop  is  sufficiently  small  this  will  be 
a  short  process.  It  may  be  hastened  by  holding  the  cover-glass  high 


32  GENERAL  BACTERIOLOGY 

over  the  flame,  but  it  should  always  be  held  in  fingers  to  prevent  over- 
heating, which  spoils  the  preparation. 

"When  the  film  is  thoroughly  dry  place  the  cover-glass  in  a  pair 
of  Cornet  or  Stewart  forceps  and  "fix"  the  bacteria  in  the  flame. 
This  is  done  by  passing  the  prepa.ra.tion 
through  the  upper  portion  of  a  gas  fla 
film  side  up.  Three  passages  should  be 

made,  each  consuming  about  one  second  forceps.  (Muir&  Ritchie). 
of  time.  The  forceps  are  now  placed  on  the  table  and  the  film  flooded 
with  one  of  the  anilin  dyes.  After  the  stain  has  acted  for  five  to 
ten  minutes  it  is  washed  off  into  a  waste  dish  with  a  stream  of  dis- 
tilled water,  and  while  the  cover-glass  is  still  wet  it  is  placed,  bac- 
teria side  down,  on  a  clean  glass  slide,  being  careful  to  avoid  air 
bubbles.  The  surplus  water  is  then  taken  up  by  means  of  a  small 
piece  of  blotting  or  filter  paper. 

The  preparation  is  now  ready  for  microscopical  examination. 
(For  directions  see  next  exercise). 

The  preparation  can  be  made  permanent  either  by  allowing  the 
water  under  the  cover-glass  to  dry  before  it  is  removed,  or  by  floating 
it  off  with  water  and  afterwards  drying.  When  dry  a  drop  of 
Canada  balsam,  dissolved  in  xylene,  is  placed  on  the  cover-glass  and 
this  is  then  lowered  on  to  the  slide  again. 

Resume. 

a.  Spread  film, 

b.  Air  dry, 

c.  Fix, 

d.  Stain, 

e.  Mount  in  water,  /          \     e.  Dry, 

f.  Examine,  3    or   v    /.  Mount  in  balsam, 

g.  Dry  and  mount  in  balsam.  (          )     g.  Examine. 

"The  great  mistake  made  by  beginners  is  to  take  too  much 
growth,"  (M.  &  K.)  and  too  large  a  drop. 

REFERENCES.  A.  159 ;  H.  80 ;  M.  &  R.  98 ;  McF.  145 ;  P.  198 ; 
P.  B.  C.  11. 

SPECIAL  DIRECTIONS. 

a.  Make  cover-glass  preparation  from  agar  streak  of  B.  subtilis 
(13)  staining  with  an  aqueous  solution  of  gentian  violet  for  five 
minutes. 


34  GENERAL  BACTERIOLOGY 

b.  Practice  making  cover-glass  preparations  by  staining  speci- 
mens from  each  of  your  cultures.  Use  Loeffler's  methylen  blue  for 
the  gelatin  and  bouillon ;  aqueous  solution  of  gentian  violet  for  agar, 
and  carbol-fuchsin  for  potato.  Examine,  mount  permanently  and 
hand  to  instructor  for  inspection. 


EXERCISE  19.     USE  OF  MICROSCOPE. 

GENERAL  DIRECTIONS.  For  bacteriological  purposes  a  microscope 
with  a  magnifying  power  of  at  least  500  diameters  is  needed.  There 
should  be  a  coarse  adjustment  (rack  and  pinion)  as  well  as  a  fine 
micrometer  screw ;  and  the  following  accessories :  Two  oculars,  one 
1  in.  (25  mm.)  and  one  2  in.  (50  mm.)  ;  three  objectives,  one  2  in. 
(16  mm.),  one  -J-  in.  (4  mm.),  or  |  in.  (3.5  mm.)  and  one  oil 
immersion  -fa  i*1-  or  TS"  in-  (2  mm.)  ;  a  triple  nose-piece,  and  an 
Abbe  substage  condenser  with  iris  diaphragm  mounting. 

In  the  use  of  the  microscope  the  following  points  should  be  noted : 

a.  LIGHT.  The  proper  angle  at  which  the  mirror  should  be 
placed  is  best  determined  by  removing  the  ocular  and  so  arranging 
the  mirror  that  the  unobstructed  light  from  the  window  covers  the 
whole  field.  The  ideal  light  is  that  from  a  white  cloud.  Direct 
sunlight  should  never  be  used. 

~b.  ABBE  CONDENSER.  The  purpose  of  the  condenser  is  to  furnish 
a  large  cone  of  light,  and  as  it  is  corrected  for  parallel  rays  the  plane 
side  of  the  mirror  should  always  be  used,  except  when  artificial  light 
is  employed.  When  highly  stained  objects  are  to  be  examined,  the 
open  diaphragm  should  be  used,  but  when  the  structural  rather  than 
the  color  picture  is  desired,  it  will  be  necessary  to  diminish  the  light 
by  closing  the  diaphragm.  When  the  high  powers  are  employed, 
raise  the  condenser  as  high  as  possible ;  for  low  powers  a  lower  posi- 
tion will  give  better  definition. 

c.  FOCUSING.     Turn  the  proper  objective  in  place  and  rack  down 
until  the  objective  nearly  touches  the  cover-glass.     This  should  be 
done  while  the  eye  is  held  at  one  side  and  directs  the  movement. 
Then  with  the  eye  at  the  tube  slowly  move  up  with  the  micrometer 
screw.     Never  rack  down  with  the  eye  at  the  tube. 

d.  USE  OF  OIL-IMMERSION.     The  oil-immersion  objective  is  indis- 
pensable to  the  proper  study  of  bacteria.     It  is  constructed  upon  the 
principle  that  a  drop  of  fluid  having  the  same  refractive  index  as  the 


36  GENERAL  BACTERIOLOGY 

objective,  prevents  the  dispersion  of  light,  thus  permitting  the  use 
of  lenses  having  a  greater  numerical  aperture  and  longer  working 
distance  for  the  same  degree  of  amplification  than  is  possible  with 
the  dry  system.  In  using  an  immersion  lens,  place  a  small  drop  of 
immersion  oil  on  the  preparation,  then  carefully  lower  the  objective 
until  it  touches  the  oil  drop  and  nearly  touches  the  cover-glass. 
Apply  eye  to  the  ocular  and  focus  upward  very  slowly  with  fine  ad- 
justment until  the  definition  is  clear.  At  the  close  of  the  day's 
work  the  oil  must  be  removed  from  the  objective  and  cover-glass. 
This  is  best  accomplished  by  wiping  them  with  a  piece  of  Japanese 
paper  made  for  the  purpose.  In  case  the  oil  should  accidentally  dry 
on  the  objective,  it  can  be  removed  by  adding  a  little  more  oil  and 
allowing  it  to  stand  for  a  few  minutes;  it  can  then  be  wiped  off 
with  paper.  If  this  method  does  not  succeed,  the  objective  should 
be  taken  to  the  instructor.  Great  care  must  be  observed  since 
solvents  of  the  oil  are  also  solvents  for  the  lens  mountings. 

KEFERENCES.     See  Gage ;  A.  199  ;  H.  118  ;  M.  &  R.  87 ;  P.  206. 

SPECIAL  DIRECTIONS.  Examine  cover-glass  preparations  made  in 
previous  exercise,  first  with  \  in.  objective,  and  then  with  the  oil- 
immersion  objective.  If  the  specimen  be  satisfactory,  sketch  as 
directed  in  next  exercise. 

EXERCISE  20.     DRAWING  BACTERIA 

GENERAL  DIRECTIONS.  In  drawing  bacteria  only  a  few  organisms 
occurring  in  the  microscopic  field  should  be  sketched,  but  these  should 
be  made  of  considerable  size  so  that  the  exact  outline  may  be  indi- 
cated. Furthermore  they  should  be  drawn  to  scale  and  individuals 
selected  to  give  range  in  form  and  size. 

To  measure  microscopic  objects  an  ocular  micrometer  is  used, 
and  the  first  step  will  be  to  determine  its  value.  Place  the  ocular 
micrometer  on  the  diaphragm  in  the  ocular,  use  a  stage  micrometer 
as  an  object  and  focus.  The  image  of  the  scale  on  the  stage  micro- 
meter will  appear  imposed  on  that  of  the  ocular  micrometer.  Make 
the  lines  of  the  two  micrometers  parallel  and  then  make  any  two 
lines  of  the  stage  micrometer  coincide  with  any  two  on  the  ocular 
micrometer,  pulling  out  the  draw-tube  if  necessary.  Divide  the 
value  of  the  included  space  or  spaces  on  the  stage  micrometer  by 
the  number  of  divisions  on  the  ocular  micrometer  required  to  in- 
clude them,  and  the  quotient  so  obtained  will  give  the  valuation  of 
the  ocular  micrometer  in  fractions  of  the  units  of  measure  of  the 
stage  micrometer  ( Gage) .  If  the  result  be  not  in  terms  of  the  micron 


38 


GENERAL  BACTERIOLOGY 


it  should  be  converted  to  such,  as  this  is  the  unit  in  micrometry. 
REFERENCES.     G.  100-108. 
SPECIAL  DIRECTIONS. 

a.  Determine  the  value  of  the  ocular  micrometer  and  fill  out 
blanks  in  following  table  : 


No    of  IMicroscope 

IMake 

Ocular  i 

n.,  or  mm. 

Objective. 

Tube  length. 

Value  of  single  di- 
vision on  scale 
in  fA. 

%in.  (16mm.) 

%in.  (4mm.) 

Oil-immersion. 

&.  Measure  the  bacteria  on  the  preparations  made  in  Exercise  18 
and  sketch  a  few  individuals  from  each. 

In  making  drawings,  represent  a  micron  by  two  and  one-half 
millimeters  on  paper.  This  will  give  a  magnification  of  2,500  diame- 
ters, represented  thus :  X  2,500. 

EXERCISE  21.  HANGING-DROP  PREPARATION. 
GENERAL  DIRECTIONS.  These  are  made  by  adding  a  small  portion 
of  bacterial  culture  from  solid  media  to  a  drop  of  water  on  a  clean 
cover-glass,  or,  in  case  of  fluid  media,  by  placing  a  small  loopful  of 
the  culture  medium  on  the  cover-glass.  A  hollow  ground  glass  slide 
having  the  rim  of  the  cavity  previously  coated  with  vaseline,  is 
inverted  and  lowered  over  the  cover-glass  enclosing  the  drop.  With 
a  careful,  quick  movement  the  preparation  is  now  brought  right 
side  up. 


2 


FIG.  12.    Hanging-drop  preparation,    a,  Hanging  drop;  &,  Vaselin. 


40  GENERAL  BACTERIOLOGY 

Instead  of  the  hollow  ground  glass-slide  an  ordinary  glass-slide 
to  which  a  small  section  of  a  glass  or  rubber  tube  has  been  cemented 
can  be  used,  and  in  some  cases  is  preferable. 

In  examining  the  preparation  under  a  microscope,  focusing  is  a 
somewhat  difficult  process  and  must  be  carried  out  with  great  care. 
Use  a  narrow  diaphragm.  Find  the  edge  of  the  drop  with  the  low 
power  (•§•  in.  objective),  adjusting  slide  so  that  edge  of  drop  passes 
through  the  center  of  the  field;  then  turn  on  the  high  power  (-J-  in. 
objective)  and  focus  without  moving  the  slide.  The  edge  of  the  drop 
is  selected  because  the  bacteria  are  here  nearest  the  cover-glass  and 
hence  more  easily  focused  upon  than  where  they  are  deeper  in  the 
drop. 

REFERENCES.     A.  204 ;  H.  114 ;  M.  &  R.  87 ;  McF.  141 ;  P.  209. 

SPECIAL  DIRECTIONS. 

a.  Make  a  hanging-drop  preparation  of  water  containing  parti- 
cles of  India  ink  or  carmine  in  suspension.  This  illustrates  molec- 
ular or  Brownian  movement. 

ft.  Make  a  preparation. using  straw  infusion  or  tartar  from  teeth 
to  note  variations  in  rate  and  character  of  vital  movement. 

c.  Make  hanging-drop  preparation  of  B.  subtilis  from  agar  or 
bouillon  (13). 

d.  Make  same  preparation  of  B.  coli  (13). 

In  cases  where  vital  movement  is  questionable,  remove  the  cover- 
glass  and  place  a  drop  of  formalin  or  chloroform  in  the  bottom  of  the 
cell ;  replace  the  cover-glass,  examine  and  note  change  in  character 
of  movement,  if  any. 


EXERCISE  22.     MICROSCOPICAL  STUDY  OF  FORM  TYPES. 

a.  Make  bouillon  and  agar  streak  cultures  of  the  following  or- 
ganisms : 

Micrococcus  (any  species). 

Sarcina  lutea  SCHROETER. 

Pseudomonas  fluorescens  (FLUEGGE)  MIG. 

Bacillus  mycoides  FLUEGGE. 

Microspira  Metschnikovi  MIG.  (or  any  vibrio). 

Spirillum  rubrum  v.  ESMARCH. 

I.  Incubate  cultures  at  28°  C.  for  24  hours. 


42  GENERAL  BACTERIOLOGY 

c.  Make  cover-glass  preparations  from  the  agar  streaks  and  stain 
with  an  aqueous  solution  of  gentian  violet  or  with  Loeffler  's  methylen 
blue. 

d.  Examine   with  the   oil-immersion   objective,   and  write   the 
names  of  the  organisms  in  their  proper  places  in  the  table  below : 


Shape  of 
organism. 

Eelative 
size. 

Name. 

Sketch. 

Spherical. 

Medium. 

Small. 

Elongated. 

Large. 

Small. 

Spiral. 

Short. 

Long. 

e.  Make  sketches  of  each  organism. 

/.  Mount  all  preparations  in  balsam  and  hand  them  to  instructor 
for  inspection. 


EXERCISE  23.  STUDY  OF  CELL  GROUPING. 

IMPRESSION  PREPARATIONS.  The  exact  relation  of  cell  to  cell  as 
they  develop  in  the  colony  can  frequently  be  determined  best  by 
studying  a  "contact  preparation"  which  is  prepared  as  follows : 

a.  Melt  a  gelatin  tube  and  slope  it,  when  solid  make  a  streak 
culture  of  B.  mycoides  and  when  growth  has  taken  place  dip  the  tube 
in  hot  water  to  loosen  gelatin,  which  is  then  slipped  out  of  the  tube. 

6.  Lower  gently  a  clean  cover-glass  over  the  surface.  Apply  a 
slight  pressure  by  tapping  glass.  Raise  cover-glass  by  one  edge, 
taking  care  that  natural  arrangements  of  adherent  bacteria  are  not 
disturbed. 

c.  Thoroughly  air  dry  the  same,  then  fix  and  stain  in  the  ordinary 
manner. 


44 


GENERAL  BACTERIOLOGY 


d.  Examine  the  thinner  layers,  noticing  the  arrangement  of  cells 
with  reference  to  each  other,  and  draw  a  sufficient  number  to  illus- 
trate this  relationship. 

HANGING-DROP  PREPARATIONS. 

a.  Make  hanging-drop  preparations  from  bouillon  cultures  pre- 
pared above  (22)  and  also  from  those  supplied. 

&.  Examine  with  oil-immersion  objective  and  assign  organisms 
to  their  proper  places,  as  determined  by  cell  grouping,  in  the  follow- 
ing scheme : 


Arrangement. 

Form. 

Name. 

Sketch. 

Isolated. 

Spheres. 

Kods. 

Spirals. 

Filaments. 

Spheres. 

Rods. 

Spirals. 

Plane  surfaces. 

Spheres. 

Eegular  masses. 

Spheres. 

Irregular  masses. 

Spheres. 

Eods. 

AGAR  HANGING-DROP  CULTURES  (Wesbrook). 
a.  Melt  a  tube  of  agar  and  cool  to  43°  C. 

Z>.  Sterilize  a  cover-glass  by  passing  it  two  or  three  times  through 
the  flame  quickly. 

c.  With  the  needle  make  a  streak  on  the  cover  glass  about  3  mm. 
long  of  B.  subtilis. 


46 


GENERAL  BACTERIOLOGY 


d.  With  the  loop  place  a  drop  of  liquid  agar  so  as  to  cover  up 
streak. 

e.  Flame  a  hollow-ground  slide  and  seal  the  cover  glass  to  it.     In- 
cubate and  later  examine  and  sketch. 

REFERENCES.  Hill,  Hanging  Block,  Jour.  Med.  Research,  1902, 
2;  202. 

EXEECISE  24.     STUDY  OF  INVOLUTION  FORMS. 

a.  Grow  Bacillus  subtilis  (EHRENB.)  MIG.  in  bouillon,  and  also 
in  water  containing  0.1%  asparagin,  10%  sugar,  and  by  means 
of  stained  cover-glass  preparations  compare  the  individual  organ- 
isms in  each  case  in  regard  to  their  form  and  size.  The  degenerated 
or  involution  forms  are  more  apparent  by  staining.  Draw  several 
cells  illustrating  a  variety  of  involution  forms. 

~b.  Examine  a  culture  of  Bacterium  diphtheriae  (LOEFFLER)  MIG. 
on  Loeffler's  blood  serum.  Read  M.  &  R.  5. 


EXERCISE  25.     STUDY  OF  ENDOSPORES. 

a.  Make  cultures  on  peptoneless  agar,  or  on  an  agar  tube  to  which 
a  few  drops  of  calcium  hydrate  have  been  added,  of  the  following 
organisms  and  incubate  at  28°  or  38°  C. : 

Bacillus  subtilis  (EHRENB.)  COHN. 

Bacterium  anthracis  (Kocn)  MIG.  (or  Bacillus  mycoides 
FLUEGGE). 

Bacillus  amylobacter  VAN  TIEGHEM  (or  any  clostridium  form). 

Bacillus  tetani  NICOLAIER  (or  any  "drumstick"  bacillus). 

ft.  When  the  cultures  are  48  hours  old  mount  films  without  stain- 
ing, examine  and  fill  out  following  table : 


Size  of  Spore. 

Position. 

Name  of  organism. 

Sketch. 

Smaller  than  di- 
ameter of  mother- 
cell. 

Median. 

Polar. 

Larger  than  diam- 
eter of  mother- 
cell. 

Median. 

Polar. 

48  GENERAL  BACTERIOLOGY 

c.  Simple  stain  for  spores. 

1.  Prepare  film  of  B.  subtilis. 

2.  Fix  by  passing  through  flame  10  or  12  times  instead  of  3  times. 
(This  prevents  the  vegetative  portion  from  taking  the  stain). 

3.  Stain  2-5  minutes  in  hot  carbol-fuchsin. 

4.  Mount  and  examine. 

d.  Double  stain  for  spores  (Hauser's  method). 

1.  Prepare  a  film  of  any  of  the  above  organisms  (providing  a 
previous  examination  has  shown  that  the  spores  are  fully  developed 
and  the  mother-cells  have  not  disintegrated). 

2.  Fix,  three  times  through  the  flame. 

3.  Stain  with  hot  (steaming)  carbol-fuchsin  for  5  minutes. 

4.  Cautiously  decolorize  with  5  per  cent,  acetic  acid  until  the 
pink  color  is  nearly  removed  from  the  film. 

5.  Wash  thoroughly  in  water. 

6.  Dry  (blot). 

7.  Stain  with  Loeffler's  methylen  blue,  3  minutes. 

8.  Mount  and  examine.     The  spores  should  appear  crimson  in 
blue  bacilli. 

KEFERENCES.  A.  171 ;  H.  98 ;  M.  &  R.  106 ;  McF.  154 ;  P.  46  & 
203 ;  P.  B.  C.  15. 

EXERCISE  26.     FLAGELLA  STAIN  (BUKGE). 

GENERAL  DIRECTIONS. 

a.  Make  an  agar  streak  of  the  organism  to  be  stained. 

I).  After  18  to  24  hours,  by  means  of  the  platinum  needle  re- 
move a  portion  of  the  growth  (being  careful  to  avoid  the  culture 
medium)  to  a  large  drop  of  tap  water  on  a  perfectly  clean  cover- 
glass  (16).  Allow  this  to  stand  5  minutes  rather  than  spread,  as 
there  is  less  danger  of  breaking  off  the  flagella. 

c.  Spread  carefully  2  or  3  loopfuls  of  this  drop  on  each  of  sev- 
eral clean  cover-glasses  and  dry  at  room  temperature. 

d.  Fix  by  passing  the  cover-glass  while  it  is  held  in  the  hand, 
(not  in  the  forceps,  as  over-heating  will  injure  the  preparation) 
through  the  top  of  the  flame. 

e.  Flood  the  cover-glasses  thus  prepared  with  the  following  solu- 
tion (mordant)  :    Liquor  ferri  sesquiMoridi  diluted  with  distilled 
water  1  :  20,  1  part ;  saturated  aqueous  solution  of  tannic  acid,  3 
parts.     This  mixture  improves  with  age  but  should  be  filtered  before 
using.     Allow  to  act  1  minute. 

f.  Wash  in  water  and  dry  between  filter  paper. 


50  GENERAL  BACTERIOLOGY 

g.  Stain  with  hot  carbol-fuchsin  for  about  one  minute. 
h.  Wash  in  water,  dry  and  mount  in  balsam. 
KEFEBENCES.     A.  174 ;  H.  100 ;  M.  &  R.  107 ;  McF.  156 ;  P.  205. 
SPECIAL   DIRECTIONS.     Stain   B.    typhosus   from   cultures   fur- 
nished, also  try  B.  coli  and  B.  subtilis. 

EXERCISE   27.     CAPSULE   STAIN    (WELCH). 
GENERAL  DIRECTIONS. 
a.  Spread  film  without  the  use  of  water, 
fc.  Air  dry. 

c.  Fix. 

d.  Apply  glacial  acetic  acid,  and  drain  it  off  immediately.     Do 
not  wash  in  water. 

e.  Apply  Ziehl's  carbol-fuchsin  which  is  to  be  renewed  several 
times  to  remove  acid. 

/.  Wash  in  1  to  2%  salt  solution. 

g.  Examine  in  salt  solution.     (Balsam  causes  capsule  to  shrink). 

REFERENCES.  A.  170 ;  H.  97  ;  M.  &  R.  106 ;  McF.  291 ;  P.  203  ; 
P.  B.  C.  13. 

SPECIAL  DIRECTIONS.  Use  pneumonic  ("rusty")  sputum,  blood 
of  rabbit  infected  with  the  Bact.  pneumoniae  or  a  milk  culture  of  a 
capsule  bearing  organism  as  Bact.  pneumonicum  (Fried.)  Mig.  or 
Bact.  capsulatum  (Stern.)  Chester. 

EXERCISE   28.     STAIN  FOR  MET  ACHROMATIC   GRANULES    (ERNST). 

a.  Stain  a  young  culture  of  an  organism  such  as  Bact.  diphtheriae 
with  Loeffler  's  methylen  blue  for  about  3  minutes. 

b.  Wash  in  water. 

o.  Treat  with  a  saturated  solution  of  Bismarck  brown  for  30  sec- 
onds. 

d.  Wash  in  water,  mount  in  water  and  examine,  or,  dry,  mount 
in  balsam  and  then  examine. 

The  granules  should  appear  blue  in  a  brown  organism. 

EXERCISE  29.     MORPHOLOGY  OF  YEASTS  AND  MOULDS  COMPARED 

WITH  BACTERIA. 

a.  Mount  some  baker's  yeast  (Sac char omyces  cerevisiae)  and 
examine  in  an  unstained  condition.  Compare:  Size;  form;  struc- 
ture and  method  of  reproduction  with  the  bacteria. 

~b.  In  same  way  examine  a  number  of  common  moulds,  e.  g. 
Mucor,  Penicillium  and  Aspergillus. 


GENERAL  BACTERIOLOGY 


EXERCISE  30.  GELATIN  PLATE  CULTURES. 

EXPLANATORY.  Plate  cultures  are  only  possible  with  the  lique- 
fiable  solid  media,  gelatin  and  agar.  In  making  them  the  bacteria 
are  mixed  with  the  medium  while  it  is  in  a  fluid  state  and  spread  out 
on  a  horizontal  surface  to  cool.  The  dilution  is  such  that  the  indi- 
viduals are  separated  from  each  other  by  several  millimeters.  In 
the  solidified  medium  the  organisms  are  fixed  and  their  growths 
result  in  the  formation  of  "colonies."  These  vary  in  size  and  ap- 
pearance according  to  the  peculiarities  of  the  organism  and  the  age 
of  the  culture,  but  are  of  the  greatest  service  in  the  study  and  identi- 
fication of  the  various  species.  These  cultures  are  prepared  as  fol- 
lows: 

GENERAL  DIRECTIONS.  Three  gelatin  tubes  are  marked  Nos.  1,  2 
and  3  and  melted  by  placing  them  in  a  water  bath  at  a  temperature 
of  42°  C.  For  this  purpose  a  small  cup  of 
water  placed  on  a  tripod  can  be  used  (Fig. 
13).  They  are  inoculated  by  introducing  the 
material  to  be  studied  into  tube  No.  1.  The 
quantity  of  this  material  varies.  The  amount 
clinging  to  the  platinum  needle  will  be  suffi- 
cient if  a  pure  culture  be  used,  while  in  other 
cases  several  loops  or  even  drops  are  necessary. 
The  inoculated  material  is  thoroughly  mixed 
with  the  gelatin  in  No.  1.  This  is  done  by 
rolling  the  tube  gently  between  the  palms  of 

FIG.  13.    Method  of  melt-  the  hands>  instead  of  shaking,  so  as  to  prevent 
ing  gelatin.  the  introduction  of  air  bubbles.     With  a  ster- 

ile loop  two  loopfuls  of  fluid  gelatin  are  now  transferred  from  No.  1 
to  No.  2,  and  mixed.     For  method  of  handling  tubes  see  Fig.  14. 

In  like  manner 
three  or  more 
loops  from  No.  2 
are  carried  over 
to  No.  3,  which 
in  turn  is  well 
mixed.  The  con- 
tents of  each  of 
the  tubes  are 
now  poured  into 

PIG.  14.    Another  method  of  holding  test-tubes.  separate     sterile 

Petri  dishes.     The  process  of  pouring  is  performed  as  follows :     The 


54 


GENERAL  BACTERIOLOGY 


Petri  dish  is  placed  on  the  desk;  the  gelatin  tube  is  taken  in  the 
right  hand,  the  cotton  plug  removed  with  the  left  hand ;  the  mouth 
of  the  tube  sterilized  by  naming  it  once  or  twice,  and,  when  the  glass 
is  cool,  the  gelatin  is 
poured  into  the  lower  half 
of  the  dish  while  the  cover 
is  slightly  raised  (Fig. 
15),  but  not  inverted  or 

T    • -.  ,,          ,    i  i         mi  FIG.  15.    Method  of  pouring  plates. 

laid    on    the     table.     The 

cover  of  the  dish  is  then  replaced,  the  test-tube  filled  with  a  solu- 
tion of  corrosive  sublimate,  and  the  cotton  plug  returned.  The 
gelatin  is  spread  over  the  entire  bottom  of  the  dish  by  tipping  it 
from  side  to  side.  It  is  then  allowed  to  harden  by  placing  the  dish  on 
the  cooling  apparatus,  or  leaving  it  on  a  horizontal  surface  at  room 
temperature.  A  simple,  inexpensive  and  effective  cooling  apparatus 

is  a  piece  of  soapstone,  such  as  is 
sold  at  hardware  stores  (Fig. 
16).  In  winter  this  can  be 
cooled  by  hanging  it  out  of 
doors,  at  other  seasons  by  im- 
mersing it  in  cold  water.  The 
three  Petri  dishes  thus  prepared 
should  be  properly  labeled  and 
placed  under  conditions  where  the  gelatin  will  remain  solid  and  yet 
growth  will  take  place.  The  temperature  of  the  laboratory  should 
not  be  allowed  to  exceed  23°  C.  or  gelatin  cultures  are  in  danger  of 
melting  while  under  examination.  Within  a  few  days  colonies  will 
make  their  appearance,  in  varying  numbers,  depending  upon  the 
dilution  used. 

Inasmuch  as  the  first  plate  is  generally  too  thickly  seeded  to  be 
of  much  service,  this  gelatin  tube  is  often  replaced  by  a  water  blank, 
which  is  treated  exactly  as  the  gelatin  tube  No.  1,  but  is  not,  of  course, 
"plated"  but  used  simply  to  dilute  the  material. 

KEFERENCES.     A.  130 ;  H.  65 ;  M.  &  R.  53 ;  McF.  199 ;  P.  224. 

SPECIAL  DIRECTIONS. 

a.  Make  three  gelatin  plate  cultures,  as  directed  above,  and  inoc- 
ulate with  B.  subtilis,  introducing  a  minute  portion  of  agar  culture 
(13)  into  tube  No.  1,  two  loops  of  No.  1  into  No.  2,  and  three  of  No. 
2  into  No.  3.  Label,  and  when  the  gelatin  has  solidified,  place  plates 
in  cool  chamber  (14). 


FIG.  16.    Soapstone  used  for  solidifying 
gelatin  in  Petri  dishes. 


56 


GENERAL  BACTERIOLOGY 


6.  Also  make  a  "blank"  plate  from  an  uninoculated  gelatin  tube, 
observing  all  precautions  to  prevent  contamination.  This  will  serve 
as  a  control  or  check  on  your  other  plates.  If  any -colonies  develop 
on  this  it  will  indicate  carelessness. 

EXERCISE  31.  AGAR  PLATE  CULTURES. 

GENERAL  DIRECTIONS.  These  are  made  in  the  same  way  as  the 
gelatin  plates  except  that  the  high  melting  point  (96°  C.)  of  agar 
makes  it  necessary  to  use  boiling  water  to  melt  it.  Inasmuch  as  the 
vitality  of  vegetative  bacteria  is  destroyed  at  a  temperature  much 
above  42°  C.  it  must  be  cooled  down  before  it  is  inoculated,  but  as 
agar  solidifies  at  39-40°  C.  it  must  not,  therefore,  be  cooled  below 
that  point.  It  is  best  to  keep  thg  melted  agar  at  about  45°  C.  for  10 
minutes  before  it  is  inoculated.  For  this  purpose  a  water-bath 
should  be  so  arranged  that  the  temperature  can  be  controlled  by 

means  of  a  thermo-regulator.     A  cheap  and 
yet  satisfactory  arrangement  is  represented 
in  Fig.  17.     Inoculate,  make  dilutions  and 
^K  g  pour  as  in  case  of  gelatin,  except  that  be- 

_\\ — I  I — I  I—I  R  fore  the  agar  is  poured,  it  is  well  to  slightly 
warm  the  Petri  dishes  by  placing  them  in 
the  incubator  at  38°  C.  for  a  few  minutes, 
otherwise  the  agar  may  solidify  in  lumps  in 
the  plate.  In  cooling,  agar  shrinks  some- 
what, and  in  doing  so  water  is  expressed 
from  the  solid  jelly.  In  the  incubator  this 
condenses  on  the  under  side  of  the  cover  of 

PIG.  17.  Water-bath  for  cool-  the  Petri  dish  to  such  an  extent  that  drops 

run  down  on  to  the  culture  surface,  thus 

causing  the  developing  superficial  colonies  to  "run."  To  obviate 
this  the  Petri  dishes,  when  placed  in  the  incubator,  should  be  in- 
verted 

REFERENCES.  A.  135 ;  H.  68 ;  M.  &  R.  57 ;  N.  285 ;  P.  225 ;  P.  B.  C. 
28. 

SPECIAL  DIRECTIONS,  a.  Make  three  agar  plates  of  B.  coli;  use 
one  loop  of  bouillon  culture  (13)  for  tube  No.  1  and  proceed  as  in  30. 
6.  Invert  and  place  in  incubator  at  28°  C. 

EXERCISE  32.  ROLL  CULTURES  (ESMARCH). 

GENERAL  DIRECTIONS.  These  are  essentially  plate  cultures  in 
which  the  medium  instead  of  being  poured  out  into  dishes  is  solidi- 


58 


GENERAL  BACTERIOLOGY 


FIG.  18.    Method  of  making  Roll-cultures.    (Abbott). 


fied  in  a  thin,  even  layer  on  the  inner  surface  of  the  test-tubes.  This 
is  best  accomplished  by  means  of  a  piece  of  ice  placed  in  a  dish  on 
a  piece  of  cloth,  by  which  it  can  be  kept  in  the  desired  position 

(Fig.  18).  A  hori- 
zontal groove  is  melt- 
ed in  the  ice  by  means 
of  a  test-tube  filled 
with  hot  water.  In 
this  groove  the  test- 
tubes,  inoculated  as 
in  case  of  plate  cul- 
tures, are  rapidly 
whirled  until  the  me- 
dium is  thoroughly 
set.  Both  agar  and 
gelatin  can  be  used, 
although  gelatin  cannot  be  used  successfully  with  those  species  which 
liquefy  this  medium.  In  the  case  of  agar  the  tubes  should  be  placed 
in  a  horizontal  position  a  few  hours  (over  night)  until  the  medium 
has  become  attached  to  the  tube :  afterwards  they  can  be  stored  in 
the  usual  receptacles  for  tube  cultures. 

REFERENCES.     A.  137 ;  H.  69  ;  M.  &  R.  56  ;  McF.  206. 
SPECIAL  DIRECTIONS,     a.  Melt  a  tube  of  gelatin  and  without  inoc- 
ulating it  practice  making  a  roll-culture  as  described  above.     Avoid 
tipping  the  tube  enough  to  get  medium  on  cotton  plug.     Remelt  and 
roll  again  and  again  until  the  knack  is  acquired. 

~b.  Make  two  roll-cultures  in  gelatin  of  E.  coli  (13) ,  using  a  water- 
blank  instead  of  gelatin  tube  No.  1. 

c.  Make  two  agar  cultures  of  B.  siibtilis  in  same  way. 

d.  Incubate  6.  in  cool  chamber,  and  c.  at  28°  C. 


EXERCISE   33.     STUDY  OF  PLATE   CULTURES. 

MACROSCOPIC.  As  the  colonies  appear,  note:  a.  form,  &.  size, 
c.  surface  elevation,  d.  consistency,  e.  color.  Both  the  surface  and 
deep  colonies  should  be  described,  as  they  are  frequently  very  differ- 
ent. Drawings  should  always  be  made  wherever  they  will  be  of 
value;  study  should  be  continued  as  long  as  changes  are  noticed. 
(See  Chapter  III.) 

MICROSCOPIC.  The  colonies  appearing  on  the  plates  are  to  be 
studied  under  a  low  power  of  the  microscope.  Use  a  -f-  in.  (16  mm.) 


60  GENERAL  BACTERIOLOGY 

objective.  The  Petri  dishes  can  be  inverted,  and  thus  avoid  the 
danger  of  exposing  the  culture  to  contamination  from  the  air  except 
with  gelatin  where  liquefying  organisms  are  present.  Observe,  a. 
structure  of  colony  as  a  whole ;  &.  character  of  margin.  (See  Chap- 
ter III.) 

SPECIAL  DIRECTIONS.  Study,  write  descriptions  and  make  draw- 
ings of  all  plate  cultures.  Use  blank  pages  for  description  and 
sketch  of  cultures. 

EXERCISE  34.     USE  OF  DECOLORIZING  AGENTS. 

Make  three  cover-glass  preparations  from  a  24  hour  old  culture 
of  B.  subtilis,  staining  them  with  an  aqueous  solution  of  gentian 
violet.  Mount  in  water  and  examine.  While  they  are  still  under 
the  microscope,  place  at  one  side  of  the  cover-glass  a  few  drops  of 
one  of  the  following  solutions,  and  by  means  of  a  strip  of  filter  paper 
at  the  opposite  side  draw  the  liquid  under  the  cover  glass  until  all 
the  color  is  removed.  In  this  way  determine  the  relative  value  of 
alcohol  (95%),  acetic  acid  (5%),  and  nitric  acid  (30%)  as  decolor- 
izing agents. 

EXERCISE    35.     GRAM'S    STAIN. 

EXPLANATORY.  This  is  a  differential  stain  and  one  of  the  most 
useful.  Some  bacteria  when  stained  by  this  method  exhibit  a  dark 
violet  color,  others  remain  perfectly  colorless,  thus  rendering  pos- 
sible the  differentiation  of  bacteria  which  are  morphologically  nearly 
or  quite  identical,  and  also  greatly  facilitating  the  demonstration  of 
certain  bacteria  in  animal  tissue.  Most  of  the  pathogenic  micrococci 
retain  the  violet  stain,  although  there  are  important  exceptions. 
The  bacilli  and  spirilla  may  or  may  not  remain  colored. 

GENERAL  DIRECTIONS. 

a.  Spread  film. 

b.  Air  dry  and  fix. 

c.  Stain  with  anilin-oil  gentian  violet  1%  minutes. 

d.  Pour  off  stain  and  without  washing. 

e.  Apply  Gram's  iodine  solution  (17,  6)  1%  minutes. 

/.  Apply  96%  alcohol  3  minutes,  or  until  drippings  do  not  stain 
white  filter  paper. 
g.  Wash  in  water. 


62  GENERAL  BACTERIOLOGY 

h.  Mount  in  water  and  examine. 

i.  Dry  and  mount  in  balsam. 

REFERENCES.     A.  169 ;  H.  89 ;  M.  &  B.  102 ;  McF.  150 ;  P.  203. 

SPECIAL  DIRECTIONS.  Stain  films  of  young  cultures  of  B.  coli 
and  B.  subtilis. 

EXERCISE  36.     TUBERCLE  STAIN   (GABBETT). 

EXPLANATORY.  All  of  the  differential  methods  of  staining  the 
tubercle  bacterium  depend  upon  the  fact  that  this  germ  is  very 
resistant  towards  the  ordinary  stains,  and,  in  order  to  be  stained  at 
all  must  be  treated  with  a  dye  containing  a  mordant  and  this  either 
allowed  to  remain  in  contact  with  the  micro-organism  several  hours 
or  be  applied  hot.  The  latter  method  is  the  quicker  and  is  usually 
employed,  although  it  does  not  give  as  good  results.  When  once 
stained  this  germ  withstands  the  effect  of  decolorizing  agents  to 
such  an  extent  that  it  is  possible  to  remove  the  dye  from  all  other 
objects  on  the  cover-glass  preparation  (as  in  sputum)  while  it  retains 
its  own  color.  The  application  of  a  second  dye,  of  a  complementary 
color,  readily  distinguishes  this  germ  from  all  others  in  the  field.  A 
few  other  bacteria  have  similar  staining  qualities.  Red  is  the  usual 
stain  and  blue  the  counter  stain.  Gabbett's  method  is  one  of  the 
simplest. 

GENERAL  DIRECTIONS. 

a.  Spread  film  (sputum  from  tuberculous  patient). 

fe.  Air  dry  and  fix. 

c.  Stain  with  hot  carbol-fuchsin  2  minutes. 

d.  Wash  in  water. 

e.  Treat  with  Gabbett's  solution  %  to  1  minute. 
/.  Wash  in  water  and  examine. 

g.  Dry  and  mount  in  balsam. 

REFERENCES.     A.  167  ;  H.  244 ;  M.  &  R.  104 ;  McF.  308 ;  P.  304. 

SPECIAL  DIRECTIONS.  Stain  three  samples  of  sputa  which  con- 
tain varying  numbers  of  the  tubercle  bacteria. 


64  GENERAL  BACTERIOLOGY 


CHAPTER  II 
PHYSIOLOGY  OF  BACTERIA 


EXERCISE  37.     PEEPAEATION  OF  SPECIAL  MEDIA. 

The  following  media  will  be  necessary  for  the  work  outlined  in 
this  chapter : 

a.  DEXTROSE  BOUILLON.  To  ordinary  bouillon  add  \%  dextrose 
(c.  P.),  tube  and  sterilize  in  steamer,  not  in  autoclave,  7  test-tubes 
and  2  fermentation  tubes. 

&.  DEXTROSE  GELATIN.  \%  dextrose  (c.  P.),  tube  and  sterilize 
in  steamer,  6  tubes. 

c.  DEXTROSE  AGAR.     \%  dextrose  (c.  P.),  tube  and  sterilize  in 
steamer,  5  tubes. 

d.  LACTOSE  AGAR.     \%   lactose    (c.  P.),  tube  and  sterilize  in 
steamer,  2  tubes. 

e.  LITMUS  SOLUTION.     To  10  gms.  of  the  dried  material  add  500 
cc.  of  distilled  water,  digest  in  a  warm  place,  decant  clear  liquid  and 
add  a  few  drops  of  nitric  acid  to  produce  a  violet  color.     (Button.) 
Place  in  flasks  or  test-tubes  and  sterilize  in  steamer  three  times,  1 
tube. 

/.  DEXTROSE-FREE  BROTH.  This  is  prepared  from  beef  by  inocu- 
lating the  meat  infusion  with  an  organism  capable  of  fermenting 
sugar,  such  as  B.  coli,  and  allowing  it  to  stand  several  hours  at  38°  C. 
(Between  &.  and  c.  Exercise  4.)  The  bouillon  is  then  prepared  in 
the  usual  manner.1 

Or  DUNHAM'S  SOLUTION. 

Sodium  chloride        0.5  gm.   )  _.  .,         .,    n  .     -,.      ,      ,    «,, 

.__.      .  f  Boil  until  all  is  dissolved,  filter, 

Peptone   (Witte)      1.    gm.    V  .,.        .  ,   , 

'     _  _  f       tube  and  sterilize,  4  tubes. 

Water  100.    gms.  ) 

g.  NITRATE  SOLUTION. 


1  Smith:  Jour.  Exp.  Med.,  1897,  2:  543. 


66  GENERAL  BACTERIOLOGY 

Sodium  chloride        0.5  gm.  ""j 

Peptone  (Merck)      1.      "      !  -r,.,,       .   ,  .,.       _  .   , 

A  _  V  Filter,  tube  and  sterilize,  3  tubes. 

Potassium  nitrate     0.2    ' 

Water  1,000.    gms.  J 

h.  LITMUS  MILK. 

1)  Freshly  separated  milk,  or  if  this  is  not  available,  new  milk 
is  placed  in  a  separatory  funnel  in  an  ice  chest  over  night  to  allow 
the  separation  of  the  cream  and  the  milk  then  drawn  off. 

2)  Litmus  solution  (e.  above)   is  then  added  until  medium  is 
faintly  blue. 

3)  Tube  and  sterilize  in  the  steamer  for  30-45  minutes  on  3  or 
4  consecutive  days.     During  the  summer  months  particularly  very 
resistant  bacterial  forms  abound  in  the  milk,  so  that  it  is  necessary 
to  increase  the  number  of  applications  or  length  of  exposure.     The 
efficiency  of  the  sterilizing  process  should  be  tested  by  placing  the 
milk  in  the  incubator  for  several  days  to  see  if  any  change  occurs, 
2  tubes. 

In  addition  to  the  above  have  15  tubes  of  bouillon  (9  to  contain 
exactly  10  cc.  for  41.  and  44.)  10  tubes  of  gelatin,  15  tubes  of  agar, 
6  water-blanks  and  5  potato  tubes. 

( If  thought  desirable  the  media  required  for  Chapters  IV.  and  V. 
[Exercise  58],  may  be  prepared  at  this  time;  this  would  then  com- 
plete all  the  media  making  required  in  Part  I. ) 

EXERCISE  38.     EFFECT  OF  REACTION  OF  MEDIA  ON  GROWTH. 

GENERAL  DIRECTIONS. 

a.  Melt  6  tubes  of  gelatin  and  add,  under  aseptic  precautions,  to 
three  of  them,  respectively,  0.1  cc.,  0.3  cc.,  and  0.5  cc.  of  a  normal 
solution  of  hydrochloric  acid,  and  to  the  other  three  the  same 
amounts  of  a  normal  sodium  hydrate  solution. 

fc.  Thoroughly  mix,  solidify  gelatin  in  ice  water  and  then  inocu- 
late (stab)  each  tube  with  the  organism  to  be  studied. 

c.  Make  a  control  culture  in  a  tube  of  neutral  gelatin. 

d.  Incubate  at  18°  C.  and  note  the  effect  of  the  chemicals  on  the 
rate,  amount  and  character  of  the  growth. 

KEFERENCES.     L.  &  N.  35  ;  McF.  41. 

SPECIAL  DIRECTIONS.     Use  B.  subtilis  and  B.  coli.    Make  sketches. 

EXERCISE  39.    EFFECT  OF  CONCENTRATION  OF  MEDIA  ON  GROWTH. 

a.  Pour  about  2  cc.  of  " condensed  milk"  into  each  of  two  sterile 
test-tubes,  dilute  one  with  five  times  the  volume  of  sterile  water. 


68  GENERAL  BACTERIOLOGY 

Z>.  Inoculate  both  with  a  pure  culture  of  B.  subtilis  and  incubate 
at  28°  C.  Explain  changes  which  occur. 

c.  Test  extract  of  beef  or  syrup  in  the  same  way. 

EXERCISE  40.    EFFECT  OF  TEMPERATURE  VARIATIONS  ON  RATE  OF 

GROWTH. 

GENERAL  DIRECTIONS. 

a.  Make  four  agar  streak  cultures  of  organism  to  be  studied. 

It.  Incubate  them  at  the  following  temperatures:  Ice  chest  (7° 
C.),  room  (20°  C.),  low  incubator  (28°  C.),  blood  heat  (38°  C.). 

c.  By  frequent  observations  as  to  luxuriance  of  growth,  deter- 
mine the  optimum  temperature  of  growth  for  each. 

KEFERENCES.     F.  73 ;  L.  &  N.  44 ;  McF.  44. 

SPECIAL  DIRECTIONS.  Use  a  mesophilic  bacterium  as  B.  coli  and 
a  psychrophilic  organism  as  Ps.  violacea. 

EXERCISE  41.     DETERMINATION  OF  THERMAL  DEATH  POINT. 

GENERAL  DIRECTIONS. 

a.  Make  a  bouillon  culture  of  the  organism  to  be  tested. 

~b.  48  hours  later  heat  a  large  water-bath  to  45°  C.  Place  in 
this,  in  close  proximity  to  a  thermometer,  5  test-tubes  (16  mm.  in 
diam.)  containing  exactly  10  cc.  of  standard  bouillon.  (Reaction 
+  1.5.) 

c.  After  15  minutes  exposure  at  this  temperature  remove  the 
cotton  plug  from  one  of  the  tubes,  inoculate  the  broth  with  three 
loopfuls  (standard  size,  12)  of  the  48  hour  old  culture  (a.),  and 
carefully  mix  by  slightly  agitating  the  tube,  without  removing  it 
from  the  bath. 

d.  After  a  further  exposure  of  10  minutes  remove  the  tube  from 
the  bath  and  place  it  immediately  in  a  vessel  of  ice  cold  water  to 
cool.     Then  incubate  at  a  temperature  favorable  to  the  development 
of  the  organism  under  observation. 

e.  Raise  the  temperature  of  the  bath  5  degrees,  i.  e.,  to  50°  C., 
inoculate  another  tube.     Keep  it  at  50°  for  10  minutes,  remove,  cool 
and  incubate. 

/.  In  the  same  manner  expose  the  organism  to  the  following 
temperatures :  55°,  60°,  and  65°  C.  for  a  period  of  10  minutes  each. 

g.  In  all  cases  incubate  at  least  a  week  and  take  as  the  thermal 
death  point  the  lowest  temperature  at  which  growth  fails  to  appear. 


70  GENERAL  BACTERIOLOGY 

(In  more  accurate  work  the  temperature  should  be  determined 
within  2°  C.) 

REFERENCES.     M.  &  R.  70 ;  McF.  246 ;  P.  146 ;  P.  B.  C.  32. 
SPECIAL  DIRECTIONS.     Use  B.  coli  or  B.  typlwsus. 

EXERCISE   42.     COMPARATIVE   EFFICIENCY   OF   DRY   AND   MOIST 

HEAT. 

GENERAL  DIRECTIONS. 

a.  Charge  a  water  blank  with  culture  of  a  spore-bearing  bacillus, 
shaking  it  well  to  break  up  the  clumps. 

&.  Sterilize  eight  cover-glasses  by  passing  them  several  times 
through  the  flame,  and  place  four  in  each  of  two  sterile  Petri  dishes. 

c.  With  a  sterile  loop  place  an  equal  quantity  of  the  bacterial 
suspension  (a.)  on  each  cover-glass,  and  dry  by  placing  Petri  dishes 
in  the  incubator  with  the  covers  slightly  raised. 

d.  When  dry  place  one  Petri  dish  in  the  dry  sterilizer  (near  the 
thermometer),  and  the  other  in  the  steamer. 

e.  Keep  both  sterilizers  at  a  temperature  of  100°  C.,  and  at  the 
end  of  5,  10,  20  and  40  minutes  respectively,  remove  one  cover-glass 
from  each  Petri,  place  it  in  a  sterile  Petri  dish  and  pour  a  tube  of 
liquefied  gelatin  or  agar  over  it.     Tip  the  dish  from  side  to  side  to 
dislodge  as  many  of  the  bacteria  as  possible  from  the  cover-glass, 
solidify  the  medium  and  incubate. 

SPECIAL  DIRECTIONS.  Use  an  old  (spore-bearing)  culture  of  B. 
subtilis.  Arrange  data  in  the  form  of  a  table. 

EXERCISE  43.     EFFECT  OF  DESICCATION. 

GENERAL  DIRECTIONS. 

a.  Prepare  five  cover-glasses  each  of  a  spore-bearing  and  a  non- 
spore-bearing  culture,  as  directed  in  42. 

&.  Place  them  in  sterile  Petri  dishes,  and  dry  in  the  incubator. 

c.  Next  morning  and  every  twenty-four  hours  later  plate  one  of 
the  cover  glasses. 

d.  In  this  way  determine  the  length  of  time  the  organism  in 
question  can  withstand  desiccation. 

REFERENCES.     F.  77 ;  L.  &  X.  40. 

SPECIAL  DIRECTIONS.  Use  a  young  culture  of  B.  coli  and  an  old 
(spore-bearing)  culture  of  B.  subtilis.  Tabulate  results. 


72  GENERAL  BACTERIOLOGY 

EXERCISE  44.     EFFECT  OF  CHEMICALS  ON  BACTERIA. 

GENERAL  DIRECTIONS. 

a.  Inoculate  three  tubes  containing  10  cc.  of  sterile  bouillon,  with 
three  loopfuls  of  a  24-hour  old  broth  culture  of  organism  to  be 
studied. 

&.  Add  0.1  cc.  of  a  5%  solution  of  carbolic  acid  to  one  tube  (No. 
1)  ;  0.6  cc.  to  another  (No.  2)  ;  and  2  cc.  to  the  third  (No.  3). 

c.  Two  hours  later  transfer  three  loopfuls  from  each  tube  to 
sterile  bouillon  and  incubate  all  of  the  tubes  at  38°  C. 

d.  The  carbolic  acid  does  not  prevent  growth  in  No.  1  or  its  sub- 
culture.    In  No.  2  no  growth,  but  abundant  in  its  sub-culture  (acts 
as  an  antiseptic ) .     In  both  No.  3  and  its  sub-culture  no  growth  ( acts 
as  a  disinfectant). 

REFERENCES.     F.  81 ;  L.  &  N.  37 ;  L.  107 ;  McF.  45. 
SPECIAL  DIRECTIONS.     Use  B.  coli. 

EXERCISE  45.  RELATION  TO  OXYGEN. 

GENERAL  DIRECTIONS. 

a.  Pour  a  tube  of  melted  agar  into  a  sterile  Petri  dish,  and  when 
the  medium  has  hardened  make  several  parallel  streaks  with  a 
platinum  loop  charged  with  an  aerobic  organism. 

Z>.  Sterilize  a  piece  of  mica  or  a  cover-glass,  by  passing  it  several 
times  through  the  flame  and  place  this  over  several  of  the  streaks. 
This  is  to  shut  out  the  air  and  should  therefore  be  in  perfect  contact 
with  the  medium. 

c.  Make  another  plate  in  the  same  way,  using  an  anaerobe. 

REFERENCES.  F.  60 ;  L.  &  N.  41 ;  L.  180 ;  M.  &  R.  19 ;  McF.  212 ; 
P.  151. 

SPECIAL  DIRECTIONS.     Use  B.  subtilis  and  an  anaerobe.     Sketch. 

EXERCISE  46.     EFFECT  OF  DIRECT  SUNLIGHT. 

GENERAL  DIRECTIONS. 

a.  Make  an  agar  plate  of  the  organism  to  be  studied  (seeding 
rather  thickly). 

&.  When  agar  has  thoroughly  set,  invert  the  Petri  and  paste  on 
under  side  a  piece  of  black  paper  from  which  has  been  cut  out  a 
number  of  letters,  e.  g.  student 's  initials. 

c.  Expose  this  dish,  paper  side  up,  to  the  direct  sunlight  for  a 
number  of  hours  (4-6). 


GENERAL  BACTERIOLOGY 


d.  Remove  the  paper  and  incubate. 
REFERENCES.     F.  71 ;  M.  &  R.  20 ;  L.  77  ;  McF.  41 ;  P.  135. 
SPECIAL  DIRECTIONS.     Use  B.  prodigiosus  (EHRENB.)  FLUEGGE. 
Sketch. 

EXERCISE  47.     DETECTION  OF  GAS   (SHAKE  CULTURE). 

GENERAL  DIRECTIONS. 

a.  Melt  a  tube  of  dextrose  agar  or  dextrose  gelatin  and  inoculate 
with  a  gas-producing  organism. 

&.  Thoroughly  mix  and  solidify  by  placing  in  ice  water. 
c.  Incubate  over  night. 

REFERENCES.     H.  70 ;  L.  &  N.  89  ;  M.  &  R.  78 ;  McF.  49  ;  P.  82. 
SPECIAL  DIRECTIONS.     Use  B.  coli;  incubate.     Make  sketch. 

EXEECISE  48.     QUANTITATIVE  ANALYSIS  OF  GAS  (FERMENTATION 

TUBE). 

GENERAL  DIRECTIONS. 

a.  Inoculate  the  open  arm  of 
a  fermentation  tube  with  a  gas- 
producing  organism. 

b.  Incubate  at  38°  C. 

c.  By   frequent   observations 
determine : 

1.  Whether      growth      takes 
place  in  the  open  or  closed  arm, 
i.  e.,  whether  it  is  aerobic  or  an- 
aerobic. 

2.  The    rapidity    and    total 

amount  of  gas  formation.     Use  Frost's  gasometer.     (Plate  I.) 

3.  Kinds  of  gas.     When  the  culture  has  ceased  producing  gas, 
completely  fill  the  open  arm  with  a  2%  solution  of  sodium  hydrate; 
place  the  thumb  over  the  mouth  of  the  tube  and  thoroughly  mix  the 
NaOH  with  the  gas  in  the  closed  arm,  then  without  removing  the 
thumb  return  the  gas  to  the  closed  arm,  remove  the  thumb,  when  the 
medium  will  rise  in  the  closed  arm  to  take  the  place  of  the  absorbed 
C02.     Measure.     The   remaining  gas   is  considered   as   hydrogen; 
bring  this  into  the  open  arm,  remove  the  thumb  and  introduce  a 
lighted  match.     Air  mixed  with   the  hydrogen   present   causes   a 
slight  explosion.     Express  the  amount  of  C02  and  H.  in  the  form 

C02 
of  a  proportion. =^     . 


FIG.  19.    Fermentation  tube,  showing 
method  of  using:  gasometer. 


76  GENERAL  BACTERIOLOGY 

REFERENCES      Smith:  Wilder  Quarter  Century  Book,  1893,  p. 
187 ;  A.  212 ;  McF.  49 ;  M.  &  R.  79 ;  P.  82. 

SPECIAL  DIRECTIONS.     Use  B.  coli;  also  try  B.  subtilis. 

EXERCISE  49.     DETECTION  OF  ACIDS  AND  ALKALIES  (WURTZ). 

GENERAL  DIRECTIONS. 

a.  Melt  a  tube  of  lactose  agar  (or  lactose  gelatin)  and  add  enough 
of  a  sterile  blue  litmus  solution  (37  e.)  to  give  it  a  distinct  color,  cool 
to  42°  C.,  inoculate  it  with  an  acid-producing  organism  and  pour  in 
the  usual  manner. 

b.  When  the  agar  has  solidified  invert  the  dish  and  place  it  in 
the  incubator. 

REFERENCE.     McF.  51. 

SPECIAL  DIRECTIONS.     Use  sewage,  putting  a  drop  in  a  water 
blank  and  using  a  loop  or  two  of  this. 

EXERCISE  50.       QUANTITATIVE  DETERMINATION  OF  ACIDS. 

GENERAL  DIRECTIONS. 

a.  Inoculate  5  test-tubes  of  dextrose  bouillon  (or  milk)  with  an 
acid-producing  organism. 

b.  Twenty-four  hours  later  remove,  with  a  sterile  pipette,  5  cc. 
of  the  medium  from  one  of  the  tubes  and  titrate  with  a  twentieth 
normal  potassium  (or  sodium)  hydrate  solution,  using  phenolphtha- 
lien  as  an  indicator. 

c.  Make  titrations  as  described  above  on  each  of  the  four  suc- 
ceeding days,  using  the  same  amount  of  culture  each  day. 

d.  Plot  the  results,  expressing  the  number  of  cc.  of  hydrate  solu- 
tion as  ordinates  and  the  daily  intervals  as  abscissae. 

SPECIAL  DIRECTIONS.     Use  B.  coli  and  incubate  at  38°  C. 

EXERCISE  51.     DETECTION  OF  NITRITES  IN  CULTURES. 

GENERAL  DIRECTIONS. 

a.  Make  a  culture  of  a  reducing  organism  in  a  test-tube  of  the 
nitrate  solution  (37  g.). 

b.  Incubate  at  28°  C.  for  1  week,  add  1  cc.  of  each  of  following 
solutions : 

1)  Sulphanilic  acid  (para-amido  benzenesulphonic  acid)  0.5  gm. 
Acetic  acid  (sp.  gr.  1.04)  150  cc. 

2)  a-amido-naphthalene  acetate.     Boil  0.1  gram  of  solid  a-amido- 
naphthalene  in  20  cc.  of  water,  filter  the  solution  through  a  plug  of 


78  GENERAL  BACTERIOLOGY 

washed  absorbent  cotton,  and  mix  the  nitrate  with  180  cc.  of  diluted 
acetic  acid.  All  water  and  vessels  used  must  be  free  from  nitrites. 
(Leffmann  and  Beam.) 

The  presence  of  a  nitrite  is  indicated  by  a  pink  color. 

c.  A  tube  of  the  original  medium  should  be  incubated  and  tested 
as  a  control. 

REFERENCES.     A.  226 ;  McF.  53. 

SPECIAL  DIRECTIONS.     Use  sewage, 

EXERCISE  52.     DETECTION  OF  AMMONIA. 

GENERAL  DIRECTIONS. 

a.  Make  bouillon  culture  and  incubate  24  to  48  hours. 

&.  Place  in  neck  of  tube  a  piece  of  filter  paper  which  has  been 
dipped  in  Nessler's  reagent  (for  formula  see  works  on  water  analy- 
sis). A  yellow  to  reddish  brown  color  indicates  the  presence  of 
ammonia. 

REFERENCE.      L.  &  N.  78. 

SPECIAL  DIRECTIONS.     Use  sewage  to  inoculate  medium. 

EXERCISE  53.     DETECTION  OF  SULPHURETTED  HYDROGEN. 

GENERAL  DIRECTIONS. 

a.  Make  a  culture  in  a  test-tube,  or  better,  in  a  flask  of  bouillon, 
and  incubate  at  38°  C. 

6.  Twenty- four  hours  later  fasten  in  the  flask,  by  means  of  the 
cotton  plug,  a  strip  of  filter  paper  moistened  with  lead  acetate. 

c.  The  presence  of  sulphuretted  hydrogen  is  indicated  by  change 
of  color  from  brownish  to  blue.  The  color  change  is  often  slight 
and  can  be  best  detected  by  frequent  observations. 

REFERENCE.     L.  &  N.  76. 

SPECIAL  DIRECTIONS.     Use  B.  coli  or  sewage. 

EXERCISE  54.     DETECTION  OF  INDOL. 

GENERAL  DIRECTIONS. 

a.  Make  two  cultures  in  tubes  of  sugar-free  broth  (or  Dunham's 
solution ) . 

5.  Five  days  later  add  a  few  drops  of  concentrated  sulphuric 
acid.  The  appearance  of  a  pink  color  indicates  that  nitroso-indol 
has  been  formed  (cholera-red  reaction).  If  the  pink  or  deep  red 
color  does  not  appear,  add  1  cc.  of  sodium  nitrite  solution  (sodium 


80  GENERAL  BACTERIOLOGY 

or  potassium  nitrite  0.02  grams  and  distilled  water  lOOce.).     The 
appearance  of  a  red  color  indicates  formation  of  indol. 

REFERENCES.     A.  223 ;  H.  21 ;  L.  &  N.  142 ;  McF.  57 ;  M.  &  R.  80. 

SPECIAL  DIRECTIONS.     Use  B.  coli  or  sewage. 

EXERCISE  55.     DETERMINATION  OF  CHEMICAL  ENZYMES  IN  CUL- 
TURES. 

GENERAL  DIRECTIONS. 

a.  Make  two  gelatin  stab  cultures  of  a  rapidly  liquefying  organ- 
ism and  incubate  several  days  or  until  the  gelatin  has  all  been  lique- 
fied and  then  add  to  each  TV  cc.  of  a  5%  solution  of  carbolic  acid  for 
each  cc.  of  medium,  shake  thoroughly  and  filter. 

5.  Pour  one  into  a  tube  of  sterile  gelatin  and  the  other  into  a 
tube  of  milk  and  note  changes. 

REFERENCE.     McF.  56. 

SPECIAL  DIRECTIONS.     Use  B.  subtilis  or  B.  prodigiosus. 

EXERCISE  56.     VARIATION  IN  ENZYME  PRODUCTION. 

Make  stab  cultures  of  Pseudomonas  aeruginosa  (SCHROETER) 
MIG.  (B.  pyocyaneus),  or  any  slow  liquefier,  in  ordinary  neutral 
gelatin  and  also  in  dextrose  gelatin.  Compare  rate  of  liquefaction 
in  each. 

EXERCISE  57.     VARIATION  IN  COLOR  PRODUCTION. 

Make  an  agar  streak  of  B.  prodigiosus.  Incubate  at  38°  C. ;  24 
hours  later  transfer  to  fresh  media.  Continue  the  process  of  daily 
transplanting  from  cultures  of  previous  day  until  chromogenic 
property  is  lost,  even  at  the  room  temperature. 


82  GENERAL  BACTERIOLOGY 


CHAPTER  III 
TAXONOMY 


In  order  to  become  acquainted  with  a  particular  organism,  to 
differentiate  it  from  its  congeners  or  to  assign  it  a  definite  place  in  a 
system  of  classification,  it  must  be  studied  under  various  conditions 
and  its  characters  determined  as  indicated  in  the  following  table. 

POINTS  TO  BE  OBSERVED  IN  THE  STUDY  OF  BACTERIA. 

The  following  scheme  gives  the  most  important  points  to  be 
noted  in  the  description  of  an  organism,  together  with  some  of  the 
more  common  descriptive  terms  suggested  by  Chester  and  others. 

MORPHOLOGICAL    CHARACTERS. 

a.  Form  and  arrangement:  Spherical,  micrococcus,  single  and 
irregularly  grouped ;  diplococcus,  streptococcus,  tetracoccus,  sarcina, 
rods,  single,  in  chains  and  in  filaments ;  spirals. 

5.  Size. 

1.  In  terms  of  the  micromillimeter ;  breadth,  average  and  ex- 
treme length. 

2.  In  terms  of  human  blood  cells. 

c.  Stain. 

1.  Aqueous  solutions:  stains  easily  or  with  difficulty;  uniformly 
or  irregularly. 

2.  Special  stains :  Gram ;  tubercle ;  etc. 

d.  Motility. 

1.  Brownian  movement. 

2.  Vital  movement:  sluggish  or  active;  rotary  or  direct;  most 
favorable  temperature :  age ;  media :  etc. 

3.  Flagella:   stained  by  Loeffler,   Bunge  or  Van  Ermengem's 
method ;  distribution :  monotrichous,  lophotrichous  or  peritrichous. 

e.  Capsule:  stained  by  Ziehl:  Gram  or  Welch's  method;  most 
favorable  conditions ;  broad  or  narrow ;  present  in  serum,  milk  or  on 
agar  streaks. 


GENERAL  BACTERIOLOGY 


83 


/.  Spores :  time  required  for  formation ;  media ;  position  in  cell, 
center  or  end;  effect  on  shape  of  cell,  clostridium,  or  drumstick; 
germination,  time,  temperature;  stain,  Hauser  or  Moeller's  method; 
temperature  limits. 

g.  Vacuoles  (plasmolysis). 

h.  Crystals. 

i.  Involution  forms. 

j.  Pleomorphism. 

1.  Effect  of  various  media. 

2.  Effect  of  reaction  of  media. 

CULTURE    CHARACTERS. 

PLATE- CULTURES  (Gelatin  and  Agar). 

I.  Surface  Colonies. 

1.  Form:  Punctiform,  too  small  to  be  denned  by  the  naked 
eye;  circular;  oval;  fusiform,  spindle-shaped,  tapering  at  each  end; 
cochleate,  twisted  like  a  snail  shell  (Fig.  20,  A)  ;  conglomerate,  an 
aggregate  of  similar  colonies  (Fig.  20,  B)  ;  ameboid,  very  irregular 
like  the  changing  forms  of  amebae  (Fig.  20,  C)  ;  rhizoid,  of  an 
irregular  branched  root-like  character  (Fig.  20,  D)  ;  floccose,  of  a 
dense  woolly  structure;  curled,  filaments  in  parallel  strands,  like 
locks  or  ringlets  (Fig.  20,  E)  ;  myceloid,  a  filamentous  colony  with 
the  radiate  character  of  a  mould  (Fig.  20,  F)  ;  filamentous,  an  irreg- 
ular mass  of  loosely  woven  filaments  (Fig.  20,  G)  ;  rosulate,  shaped 
like  a  rosette. 


FIG.  20.  Types  of  Colonies.  A.  Cochleate  (B.  coli,  abnormal  form).  B.  Conglomerate 
(B.  Zopfii).  C.  Ameboid  (B.  Vulgatus).  D.  Rhizoid  (B.  mycoides).  E.  Curled  (B.  an- 
tnracis).  F.  Myceloid  (B.  radiatus).  G.  Filamentous. 


84  GENERAL  BACTERIOLOGY 

2.  Size  expressed  in  millimeters. 

3.  Surface  elevation:     Flat,  thin  spreading  over  the  surface 
(Fig.  21,  a)  ;  effused,  spreading  over  the  surface  as  a  thin  veilly 
layer,  more  delicate  than  the  preceding;  raised,  thick  growth,  with 

abrupt,  terraced  edges  (Fig.  21, 
b)  ;  convex,  surface  segment  of  a 
circle,  but  very  flatly  convex 
(Fig.  21,  c)  ;  pulvinate,  surface 
segment  of  a  circle,  but  decided- 
ly convex  (Fig.  21,  d)  ;  capitate, 
hemispherical  (Fig.  21,  e)  ;  urn- 
J  bilicate,  shaped  like  a  navel  (Fig. 

FIG.  21    Surface  Elevations  of  Growths.   21,  f )  ;  umbonate,  bearing  a  knob 

a.  Flat:  &,  Raised;  c,  Convex;  d,  Pulvinate;  '  ' 

e,  Capitate;  f,  Umbilicate ;  fir,  Umbonate.  in  the  Center  (Fig.  21,  g). 

4.  Topography  of  surface:     Smooth,  surface  even  without  any 
of  the  following  distinctive  characters ;  alveolate,  marked  by  depres- 
sions separated  by  thin  walls  so  as  to  resemble  a  honey  comb ;  punc- 
tate, dotted  with  punctures  like  pin-pricks ;  bullate,  like  a  blistered 
surface,  rising  in  convex  prominences,  rather  coarse ;  vesicular,  more 
or  less  covered  with  minute  vesicles  due  to  gas  formation,  more 
minute  than  bullate ;  verrucose,  wart-like,  bearing  wart-like  promi- 
nences ;  squamose,  covered  with  scales ;  echinate,  beset  with  pointed 
prominences ;  papillate,  beset  with  nipple  or  mamma-like  processes ; 
rugose,  short,  irregular  folds  due  to  shrinkage ;  contoured,  an  irreg- 
ular but  smoothly  undulating  surface  like  the  surface  of  a  relief 
map ;  rimose,  abounding  in  chinks,  clefts,  or  cracks. 

5.  Microscopic  structure. 

A.  Colony  as  whole:  Power  of  refraction,  weak  or  strong; 
amorphous,  without  definite  structure;  hyaline,  colorless  or  clear; 
homogenous,  structure  uniform  throughout;  areolate,  divided  into 
rather  irregular  or  angular  spaces  by  more  or  less  definite  boun- 
daries (Fig.  22,  1)  ;  granular,  finely  or  coarsely;  grumose,  clotted 
appearance,  particles  in  clustered  grains  (Fig.  22,  2)  ;  moruloid, 
having  the  character  of  a  morula  divided  into  more  or  less  regular 
segments  (Fig.  22,  3)  ;  clouded,  having  a  pale  ground  with  ill-defined 
patches  of  deeper  tint  (Fig.  22,  4)  ;  gyrose,  marked  by  wavy  lines 
indefinitely  placed  (Fig.  22,  5)  ;  rivulose,  marked  by  lines  like  the 
rivers  of  a  map ;  rimose,  showing  chinks,  cracks  or  clefts ;  marmo- 
rated,  showing  faint,  irregular  stripes,  or  traversed  by  vein-like 
markings  as  in  marble  (Fig.  22,  6)  ;  reticulated,  in  the  form  of  a 


GENERAL  BACTERIOLOGY 


85 


network,  like  the  vein  of  a  leaf  (Fig.  22,  7)  ;  filamentous,  floccose,  or 
curled,  as  defined  under  1  above. 


FIG.  22.  Microscopic  Structure  of  Colonies.  A,  Colony  as  a  whole.  B,  Edge  of  Colony. 
1,  Areolate;  2,  Grumose;  3,  Moruloid;  4,  Clouded;  5,  Gyrose;  6,  Marmorated;  7,  Recticu- 
Jate;  8,  Repand;  9.  Lobate;  10,  Erose;  11,  Auriculate;  12,  Lacerate;  13.  Fimbricate;  14, 
Ciliate. 

B.  Edge  of  colonies:  entire,  without  toothing  or  division; 
undulate,  wavy;  repand,  like  the  border  of  an  open  umbrella  (Fig. 
22,  8)  ;  lobate,  (Fig.  22,  9)  ;  erose,  as  if  gnawed,  irregularly  toothed 
(Fig.  22,  10)  ;  auriculate,  with  ear-like  lobes  (Fig.  22,  11)  ;  lacerate, 
irregularly  cleft,  as  if  torn  (Fig.  22,  12)  ;  -/imbricate,  fringed  (Fig. 
22,  13)  ;  ciliate,  hair-like  extensions,  radially  placed  (Fig.  22,  14)  ; 
filamentous,  (Fig.  20,  G)  ;  curled,  (Fig.  20,  E). 

6.  Color  (to  be  determined  for  both  transmitted  and  reflected 
light)  :  transparent;  vitreous,  transparent  and  colorless;  oleaginous, 
transparent  and  yellow,  olive  to  linseed  oil  colored ;  resinous,  trans- 
parent and  brown,  varnish  or  resin  colored :  translucent;  paraffinous, 
translucent  and  white,  porcelanous ;  opalescent,  translucent,  grayish- 
white  by  reflected  light,  smoky-brown  by  transmitted  light ;  nacreous, 
translucent,  grayish- white  with  pearly  lustre;  sebaceous,  translu- 
cent, yellowish  or  grayish  white,  tallowy ;  butyrous,  translucent  and 
yellow;  ceraceous,  translucent  and  wax  colored;  opaque;  cretaceous, 
opaque  and  white;   chalky,  dull  without  lustre;  glossy,  shining; 
fluorescent;  iridescent. 

7.  Consistency:     hard,  friable;  soft;  viscid. 

8.  Changes  in  medium:     Liquefaction  (gelatin),  shape  of  liqui- 
fied area,  character  of  the  fluid,  membrane  and  sediment  see  under 
Bouillon  below ;  color;  odor;  consistency. 

II.  Deep  Colonies. 

1.  Form.     2.  Size.     3.  Character    of    surface.     4.  Microscopic 
structure.     5.  Consistency.     6.  Changes  in  medium.     Same  as  sur- 
face colonies. 
STAB  CULTURES  (Gelatin  or  Agar). 

I.  Non-liquefying. 


86 


GENERAL  BACTERIOLOGY 


1.  Line  of  puncture :     filiform,  uniform  growth  without  any  spe- 
cial characters  (Fig.  23,  1)  ;  nodose,  consisting  of  closely  aggregated 
colonies;  beaded,  loosely  placed  or  disjointed  colonies  (Fig.  23,  2)  ; 
papillate,  covered  with  papillae;  echinulate,  minutely  prickly  (Fig. 
23,  3)  ;  villous,  beset  with  undivided  hair-like  extensions  (Fig.  23, 
4)  ;  plumose,  a  delicate  feathery  growth;  arborescent,  beset  with 
branched  hair-like  extensions  (Fig.  23,  5). 

2.  Surface  growth.     Same  as  for  plate  cultures. 


B 


FIG.  23.  Types  of  Growth  in  Stab  Cultures.  A,  Non-liquefying:  1,  Filiform  (B.  coli); 
2.  Beaded  (Str.  pyogenes);  3,  Echinate  (Bact.  acidi-lactici) ;  4,  Villous  (Bact.  murisepti- 
cum);  5,  Arborescent  (B.  mycoides).  B,  Liquefying:  6,  Crateriform  (B.  vulgais,  24 
hours);  7,  Napiform  (B.  subtilis,  48  hours);  8,  Infundibuliform  (B.  prodigiosus);  9,  Sac- 
cate (Msp.  Finkleri):  10,  Stratiform  (Ps.  fluorescens). 

II.  Liquefying. 

1.  Shape  of  liquefied  area:     crateriform,  saucer  shaped   (Fig. 
23,  6)  ;  napiform,  outline  of  a  turnip  (Fig.  23,7)  ;  infundibuliform, 
shape  of  a  funnel,  conical  (Fig.  23,  8)  ;  saccate,  shape  of  an  elon- 
gated sac   (Fig.  23,  9)  ;  stratiform,  liquefaction  extending  to  the 
walls  of  the  tube  and  then  downward  horizontally  (Fig.  23,  10). 

2.  Condition  of  fluid :     See  Bouillon  below. 

STREAK  CULTURES  (Gelatin,  Agar,  Potato  or  Blood  serum). 

1.  Form:     filiform  (Fig.  24,  1)  ;  nodose;  beaded  (Fig.  24,  3) ; 


GENERAL  BACTERIOLOGY 


papillate;  echinulate  (Fig.  24,  2)  ;  effused  (Fig.  24,  4) ;  villous; 
plumose;  arborescent  (Fig.  24,  5). 


FIG.  24.    Types  of  Streak  Cultures:    1,  Filiform  (B.  coli);  2,  Echinulate  (Bact.  acidi- 
lactici);  3,  Beaded  (Str.  pyogenes);  4,  Effuse  (B.  vulgaris) ;  5,  Arborescent  (B.mycoides). 

2.  Size ;  in  millimeters. 

3.  Surface  elevation. 

4.  Topography  of  surface. 

5.  Color. 

6.  Consistency. 

7.  Changes  in  medium. 


>-  Same  as  plate  cultures. 


BOUILLON  CULTURES. 

1.  Condition  of  fluid :     clear;  clouded,  degree  of,  does  or  does  not 
clear  on  standing. 

2.  Membrane:     when  formed;  color;  consistency;  structure. 

3.  Sediment :     amount;  color;  character;  whether  compact  or 
flocculent;  on  agitation  appears  granular,  flaky  or  viscid. 

4.  Reaction. 

MILK  CULTURES. 
I.  Curd  formed: 

1.  Time  required  to  curdle. 

2.  Character  of  curd:     hard  or  soft1;  massed  or  in  fragments; 
changed  or  not  on  boiling. 

3.  Whey :     amount;  transparent  or  turbid. 

4.  Reaction :     effect  on  litmus. 

5.  Digestion:     time  required;  solution  complete  or  incomplete; 
reaction;  character  of  solution,  clear,  or  cloudy. 

6.  Gas  bubbles. 

7.  Odor. 

II.  Digestion  without  formation  of  curd. 

III.  No  visible  change  even  after  boiling. 


88  GENERAL  BACTERIOLOGY 

PHYSIOLOGICAL  CHARACTERS. 

a.  Effect  of  desiccation. 

fe.  Relation  to  temperature:  minimum;  optimum;  maximum; 
thermal  death  point. 

c.  Relation  to  oxygen :  under  mica  plate ;  in  hydrogen  or  nitrogen. 

d.  Relation  to  light,  Buchner's  Experiment  (46). 

e.  Relation  to  antiseptics  and  disinfectants. 

/.  Pigment  production :  relation  of  development  to  oxygen ;  rela- 
tion of  development  to  character  of  medium ;  changes  produced  by 
alkali  and  acid ;  solubility ;  spectrum  analysis. 

g.  Gas  production :  rate,  quantity  and  formula  produced  in  dex- 
trose, lactose,  and  saccharose  media. 

h.  Acid  and  alkali  production:  carbohydrates  present;  carbo- 
hydrates absent. 

i.  Relation  of  growth  to  acidity  and  alkalinity  of  medium ;  growth 
in  1,  2,  3  and  4%  alkali ;  growth  in  1,  2,  3,  4  and  5%  acid. 

j.  Reduction  of  nitrates :  to  nitrites ;  to  ammonia.    ' 

Jc.  Production  of  sulphuretted  hydrogen. 

Z.  Production  of  indol  in  sugar-free  bouillon. 

m.  Enzyme  production ;  proteolytic ;  diastatic. 

n.  Characteristic  odor. 

o.  Pathogenesis : 

1.  Modes  of  inoculation  by  which  its  pathogenic  properties  are 

demonstrated. 

2.  Quantity  of  material  required. 

3.  Duration  of  the  disease  and  its  symptoms. 

4.  Lesions  produced  and  the  distribution  of  the  bacteria  in 

the  inoculated  animals. 

5.  Which  animals  are  susceptible  and  which  are  immune. 

6.  Variations  in  virulence  and  the  probable  causes  to  which 

they  are  due. 

7.  Detection  of  toxic  or  immunizing  products  of  growth. 

8.  Agglutinating  properties  of  serum  of  immune  animals. 

(Widal  reaction.) 

9.  Lysogenic    properties    of    serum    of    immune    animals. 

( Pf  eiff er  's  phenomenon. ) 

REFERENCES.  Chester,  Reports  Delaware  Experiment  Station, 
1897,  1898  and  1899;  A.  227;  C.  17;  H.  105;  P.  B.  C.  (Cheesman's 
Charts)  ;  Kendall,  Rept.  Am.  Pub.  H.  Assn.,  28:  481. 


GENERAL  BACTERIOLOGY 


89 


MIGULA'S  SYSTEM  OF  CLASSIFICATION. 

I.  Cells  globose  in  a  free  state,  not 
elongated  in  any  direction  be- 
fore division  into  1,  2,  or  3 
planes.  COCCACEAE  ZOPF  emend.  MIG. 

A.  Cells   without   organs   of   mo- 
tion. 

a.  Division  in  one  plane, 
6.  Division  in  two  planes, 


c.  Division  in  three  planes, 


1.  Streptococcus  BILLROTH. 

2.  Micrococcus   (HALLIER  ) 

COHN. 

3.  Sarcina  GOODSIR. 

4.  Planococcus  MIGULA. 

5.  Planosarcina  MIGULA. 
II.  Cells      cylindrical,      longer      or 

shorter,  and  only  divided  in 
one   plane,   and   elongated  to 
twice   the   normal   length   be- 
fore the  division. 
(1)   Cells  straight,  rod-shaped 
without     sheath,     non- 
motile     or     motile     by 
means  of  flagella.  BACTERIACEAE  MIGULA. 

A.  Cells    without    organs    of 

motion, 

B.  Cells   with   organs   of  mo- 

tion (flagella). 
a.  Flagella  distributed  over 

the  whole  body, 
6.  Flagella  polar, 
|(2)   Cells     crooked,     without 

sheath.  SPIRILLACEAE  MIGULA. 

X  Cells  rigid,  not  snake-like 
or  flexuous. 

a.  Cells  without  organs  of 

motion  (flagella),       -    9.  Spirosoma  MIGULA. 

b.  Cells     with     organs     of 

motion  (flagella). 

1.  Cell  with  1,  very  rare- 
ly 2-3  polar  flagella,    10.  Microspira  SCHROETER. 


-    6.  Bacterium  EHRENB. 


7.  Bacillus  COHN. 

8.  Pseudomonas  MIGULA. 


90 


GENERAL  BACTERIOLOGY 


2.  Cells  with  polar  fla- 

gella- tufts,      -        -11.  Spirillum  EHRENB. 

B.  Cells  flexuous,    -  -    12.  Spirochaeta  EHRENB. 

(3)   Cells  inclosed  in  a  sheath.  CHLAMYDOBACTERIACEAE 

MlGULA. 

A.  Cell  contents  without  gran- 
ules of  sulphur. 

a.  Cell  threads  unbranched. 
1).  Cell  division  always 

only  in  one  plane,    -    13.  Streptothrix  COHN. 

2 ) .  Cell  division  in  three 
planes  previous  to 
the  formation  of  co- 
nidia. 

i).  Cells  surrounded 
by  very  delicate, 
scarcely  visible 
sheath  (marine). 


ii).  Sheath  clearly 
visible  ( fresh  wa- 
ter), - 

&.  Cell  threads  branched, 

B.  Cell  contents  containing  sul- 
phur granules. 


14.  Phragmidiothrix   ENG- 
LER. 


15.  Crenothrix  COHN. 

16.  Cladothrix  COHN. 

17.  Thiothrix  WINOGRAD- 

SKY. 


(4)  Cells  destitute  of  a  sheath, 
united  into  threads  mo- 
tile by  means  of  an  un- 
dulating membrane.  BEGGIATOACEAE  TREVISAN. 

Only  one  genus,      -        -        -    18.  Beggiatoa  TREVISAN. 


GENERAL  BACTERIOLOGY  91 

BACTERIA  ARRANGED  IN  CLASSES  AND  GROUPS. 

Saprophilic  Class: 

Bacillus  vulgatus  Trevisan. 
Bacillus  subtilis  (Ehrenb.)  Cohn. 

Chromogenic  Class : 

Bacillus  prodigiosus  (Ehrenb.)  Fluegge. 

Zymogenic  Class : 

Bacterium  acidi-lactici  Hueppe. 

Saprogenic  Class : 

Bacillus  vulgar  is  (Hauser)  Mig. . 
Bacillus  Zopfii  (Kurth)  Mig. 

Phosphorescent  Class : 

Bacterium  phosphorescens  (Cohn)  Fischer. 

Pathogenic  Aerobes. 

Erysipelas  Group : 

Streptococcus  erysipelatos  Fehleisen. 

Pus  Coccus  Group : 

Micrococcus  pyogenes  var.  albus  (Rosenbach)  L.  &  N. 
Micrococcus  pyogenes  var.  aureus  (Rosenbach)  L.  &  No 

Malta  Fever  Group : 

Micrococcus  melitensis  Bruce. 
Diplococcus  Group : 

Micrococcus  gonorrhoeas  (Baum)  Fluegge. 

Micrococcus  Weichselbaumii  (Trevisan). 

Sarcina  Group : 

Sarcina  tetragena  (Gaffky)  Mig. 

Anthrax  Group : 

Bacterium  anthracis  (Koch)  Mig. 

Friedlander  Group: 

Bacterium  pneumonicum  (Fried.)  Mig. 
Bacterium  aerogenes  (Esch.)  Mig. 
Bacterium  capsulatum  (Sternberg)  Chester. 

Swine  Plague  Group : 

Bacterium  choleras  (Zopf )  Kitt. 
Bacterium  bovisepticum  (Kruse)  Mig. 

Glanders  Group: 

Bacterium  mallei  (Loeffler)  Mig. 
Bacterium  rhusiopathiag  (Kitt)  Mig. 


92  GENERAL  BACTERIOLOGY 

Diphtheria  Group : 

Bacterium  diphtheriae  (Loeffler)  Mig. 

Bacterium  pseudodiphtheriticum  (Loeffler)  Mig. 
Pneumonia  Group : 

Bacterium  pneumonias  (Weichsel.)  Mig. 
Influenza  Group  : 

Bacterium  influenzas  (Pfeiffer)  Lehm.  and  Neum. 
Tubercle  Group : 

Bacterium  tuberculosis  (Koch)  Mig. 

Bacterium  tuberculosis  var.  avium  (Kruse)  Mig. 
Colon  Group : 

Bacillus  coli  (Escherich)  Mig. 

Bacillus  enteritidis  Gaertner. 
Hog  Cholera  Group : 

Bacillus  Salmonii  (Trevisan)  Chester. 

Bacillus  icteroides  Sanarelli. 
Typhoid  Group : 

Bacillus  typhosus  Zopf . 
Bacillus  dysenteriae  Shiga. 

Bacillus  pestis  Lehmann  and  Neumann. 
Pseudomonas  Group : 

Pseudomonas  aeruginosa  (Schroeter)  Mig. 

Cholera  Group : 

Microspira  comma  (Koch)  Schroeter. 

Microspira  Metschnikovi  (Gamaleia)  Mig. 

Microspira  Schuylkilliensis  (Abbott)  Chester. 
Streptothrix  Group : 

Streptothrix  bovis  (Harz)  Chester. 

Streptothrix  Madurae  Vincent. 

Pathogenic  Anaerobes. 

Emphysema  Group: 

Bacterium  Welchii  Mig. 

(Edema  Group : 

Bacillus  Feseri  (Trevisan)  Chester. 
Bacillus  edematis  Zopf. 
Bacillus  botulinus  v.  Ermengem. 

Tetanus  Group : 

Bacillus  tetani  Nicolaier. 


GENERAL  BACTERIOLOGY  93 


CHAPTER  IV 

SYSTEMATIC  STUDY  OF  REPRESENTATIVE 
NON-PATHOGENIC  BACTERIA 


In  making  a  systematic  study  of  a  bacterium  it  is  necessary  to 
determine  as  many  as  possible  of  the  points  indicated  in  the  previous 
chapter  (III.)  ;  and  in  the  laboratory  this  becomes  a  regular  rou- 
tine procedure— in  the  study  of  each  germ.  The  organism  is  first 
inoculated  into  a  number  of  the  standard  media.  These  cultures  are 
frequently  spoken  of  as  a  "set  of  cultures"  and  are  usually  com- 
posed of  the  following :  Gelatin  and  agar  plates,  a  gelatin  stab,  agar 
and  potato  streaks,  a  bouillon  culture  (or  Dunham's  sol.),  a  milk 
culture  and  a  dextrose  gelatin  or  agar  stab  (or  shake  culture). 
These  cultures  are  then  incubated  at  the  proper  temperature  for  24 
hours.  They  are  then  examined,  described  and  sketched.  At  the 
same  time  three  cover-glass  preparations  are  made,  one  each  from 
the  agar,  bouillon  and  gelatin  cultures  and  stained  with  the  follow- 
ing dyes:  agar  with  an  aqueous  solution,  bouillon  with  Loeffler's 
methylen  blue,  and  the  gelatin  by  Gram's  method.  The  bouillon 
culture  is  also  examined  in  a  hanging-drop  for  motility  and  the  milk 
culture  for  capsules.  From  these  microscopical  preparations  the 
morphological  characters  can  usually  be  determined.  The  cultures 
are  again  placed  in  the  incubator  and  24  hours  later  (48  hours  after 
inoculation)  are  again  examined  and  any  changes  are  noted  and 
sketched.  The  cultures  are  now  usually  kept  at  the  temperature 
of  the  room  for  about  one  week  and  then  examined  for  the  last 
time. 

If  the  organism  produces  gas  in  dextrose  media,  fermentation 
tubes  should  be  inoculated  and  the  rate,  amount  and  formula  of  the 
gas  determined. 

The  descriptions  and  sketches  are  conveniently  made  on  the 
charts  provided  on  the  following  pages. 


94  GENERAL  BACTERIOLOGY 

EXERCISE  58.     PREPARATION  OF  SPECIAL  MEDIA. 
Tube  and  sterilize  the  following  media  for  work  in  Chapters  IV, 
and  V. : 

80  tubes  of  ordinary  or  nutrient  agar. 

2  tubes  of  lactose  agar. 
10  tubes  of  dextrose  agar  or  gelatin. 
20  tubes  of  gelatin. 
10  tubes  of  bouillon. 

10  fermentation  tubes  of  dextrose  bouillon. 
10  tubes  of  potato. 
10  tubes  of  milk. 

10  tubes  of  sugar-free  bouillon,  or  Dunham 's  solution, 
10  water-blanks. 


EXERCISE  59.     SAPEOPHILIC  CLASS.  95 

Bacillus  vulgatus  Trevisan. 

SYNONYMS.  Bacillus  mesentericus  vulgatus  Fluegge ;  Potato 
bacillus. 

EXPLANATORY.  This  is  a  widely  distributed  organism  which  was 
first  described  by  Fluegge  in  1886.  Its  spores  are  very  resistant  and 
can  almost  invariably  be  found  on  potatoes.  It  can  usually  be  ob- 
tained by  boiling  potatoes  for  a  half  an  hour,  halving  them  and  incu- 
bating in  a  sterile  moist  chamber. 

REFERENCES.  Fluegge :  Die  Mikroorganismen,  1886  ;  C.  271 ;  L. 
&  N.  323 ;  Mig.  2 :  556. 


MORPHOLOGICAL  CHARACTERS  : 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


SKETCHES. 


&.  Agar 


c.  Gelatin. 


d.  Other  media. 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 


5.    SPORES:. 


6.    SPECIAL,  CHARACTERS: — 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism 


96  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -j- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48hoursat...  ...°C.  6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  :   (b)  Deep  Colonies. 


Sketches. 


°c 

«c. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum  j 


BACILLUS  VULGATUS 


97 


Gelatin  Stab:    Grown24hours  at °C 


? 


48  hours  at , 


6  days  at oC, 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at °C.  6  days  at °C. 

Potato:    Grown  24  hours  at , °C. 

b3 

I 

48  hours  at °C.  6  days  at °C. 

!  ' 

Bouillon:    Grown  24  hours  at °C.  Q 

o 

t§ 

I  \          ) 
48  hours  at ,°C.  6  days  at °C. 


98  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture : 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION:... 


litmus  milk 


7.  REDUCTION  OF  NITRATES:. 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


SAPROPHILIC  CLASS— CONTINUED. 


99 


Bacillus  subtilis  (Ehrenb.)  Cohn. 
SYNONYMS.     Vibrio  subtilis  Ehrenberg ;  Hay  bacillus. 

EXPLANATORY.  This  is  a  well-known  and  widely  distributed  or- 
ganism. First  described  by  Cohn  in  1872.  It  is  almost  invariably 
found  on  hay,  hence  the  common  name.  Its  spores,  like  those  of  the 
''potato  bacillus,"  are  very  resistant  to  heat.  A  pure  culture  can 
usually  be  obtained  by  making  an  infusion  of  hay  or  straw  and 
heating  it  to  80°  C.  for  ten  minutes. 

REFERENCES.  F.  Cohn,  Beitraege  Zur  Biologic,  Bd.  I,  1872, 
Heft  2,  p.  175  ;  C.  276 ;  L.  170 ;  L.  &  N.  317 ;  Mig.  2  :  515. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE:  

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain  

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagrella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


100  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -j-   or  — 


Gelatin  plate:    Grown  24  hours  at 'C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media1.    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS;  SUBTILIS  \ '  V ' i  '*'. :  5 :  f  1 0 L 

Gelatin  Stab:    Grown  2A  hours  at °C.  Q  Q 

o  o 

ti 

cc 
h 

48  hours  at °C.  6  days  at 0C. 

Agar  Streak:     Grown  24  hours  at *C=  Q  Q 

'     /\ 
/     \ 

c,  , 

2  £ 

§  I  § 

,8 

/I      K 
\ 

48  hours  at °C.  6  days  at °C. 

Potato:     Grown  24  hours  at   °C.  jj  |  Q 

/\ 

I 

A^,^ 

48  hours  at °C.  6  days  at °C 

Bouillon:    Grown  24  hours  at °C.  ^  Q 


48  hours  at °C. 


6  days  at °C. 


?         »    *  »« 

rs  -O   -i-T'i  •-•-,*,•»•"     -  n-r  i-       »   "i1-  o  v 
-1  ".-">  ?"  -j  v-   -l  "  i'1      »  »  *="*  ">      -"•    » 

102  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: ,. 

optimum °C.;  limits to °C.; 

thermal  death-point °C.:  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3  RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H :  CO2:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: . . .  . ,  


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites  to  ammonia... 

8.  INDOL  PRODUCTION 

48  hours days 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) ; 


EXERCISE  60.     C1IROMOGKENIC  CLASS.  1Q3 

Bacillus  prodigiosus  (Ehrenb.)  Fluegge. 

SYNONYMS.     Monas  prodigiosa  Ehrenb.;  M.  prodigiosus  Cohn. 

EXPLANATORY.  This  organism  was  first  described  by  Ehrenberg.  It  is 
the  oldest  known  chromogenic  bacterium.  It  is  very  commonly  found  in  the 
air  of  Europe  and  has  a  very  interesting  history  on  account  of  its  casual 
relation  to  bread  epidemics — ''bloody  bread,"  "bleeding  host,"  etc.  It 
occurs  spontaneously  in  this  country.  It  is  slightly  pathogenic.  Introduced 
intraperitoneally  into  guinea  pigs  in  large  quantities  it  produces  death.  In- 
oculated into  animals  naturally  immune  to  malignant  oedema  it  renders  them 
susceptible.  Rabbits  inoculated  with  anthrax  are  protected  by  a  subsequent 
inoculation  with  this  organism.  It  is  grown  with  the  streptococcus  of  erysip- 
elas to  produce  Coley's  Fluid  for  treatment  of  inoperable  malignant  tumors. 

REFERENCES.  Ehrenberg,  Verhandlunger  der  Berliner  Akademie,  1839;  C. 
258;  L.  137;  L.  &  N.  272;  Mig.  2:  845. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


b.  Agar. 


c.  Gelatin. 


d.  Other  media. , 


2.    SIZE:  

3'.    STAINING  POWERS  : 

a    Aqueous  gentian- violet.. 

&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.    MOTILITY  : 

a.  Character  of  movement. . 
6.  Flagella  stain 


5.    SPORES:. 


SPECIAL  CHARACTERS  : 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles.. 
*?.  Pleomorphism . . 


104  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller 's  scale)  -j- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at  

...on 

«c. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  PRODIGIOSUS 


105 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at 0C. 


Agar  Streak:    Grown  24  hours  at °C. 


D 


/ 


48  hours  at 


6  days  at °C. 


Potato :    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °0. 


48  hours  at *C. 


Gdaysat ° 


106  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: ,. 

optimum °C. ;  limits to °C. : 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3-    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development :  24  hours per  cent. ,  48  hours per  cent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : : , 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI    PRODUCTION: 


litmus  milk . 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia... 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENEYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . 


GENERAL  BACTERIOLOGY 


107 


EXERCISE  61.     VARIETY  OF  PIGMENTS. 

Make  agar  or  potato  streak  cultures  of  the  following  organisms,  incubate 
at  28°  C.,  study,  describe  and  sketch. 


3  TO  6  DAYS 


SKETCHES 


Bacillus 
indicus 
or 


Sarcina 
aurantiaca 
or 


Sarcina 
lutea 
or 


Pseudomonas 
fluorescens 
(B.  fluorescens) 


Pseudomonas 
aeruginosa 
(B.  pyocyaneus) 


Pseudomonas 

violacea 

or 


108  GENERAL  BACTERIOLOGY 

EXERCISE   62.     SEPARATION  OF   BACTERIAL   COLORING   MATTER. 

a.  Make  four  agar  streaks  of  Bacillus  prodigiosus,  which  are  to 
be  kept  in  the  dark  until  the  coloring  matter  is  well  formed. 

&.  Add  about  10  cc.  of  ether  to  each  tube  and  shake  vigorously 
until  the  red  pigment  has  all  been  dissolved  out. 

c.  Pour  into  a  large  test-tube  and  allow  to  stand  over  night  in 
the  dark,  then  pipette  off  the  colored  portion. 

d.  Divide  this  into  four  parts  and  treat  them  as  follows : 

1.  Evaporate  on  glass  slide  and  examine  crystals  formed 

under  microscope. 

2.  Add  a  few  drops  of  hydrochloric  acid,  drop  by  drop 

3.  Add  a  few  drops  of  sodium  hydroxide. 

4.  Stand  in  direct  sunlight. 


EXERCISE  63.     ZYMOGENIC  CLASS.  109 

Bacterium  acidi-lactici  Zopf. 

COMMON  NAME.     Lactic  acid  bacillus. 

EXPLANATORY.  This  organism  may  be  taken  as  a  type  of  the 
bacteria  causing  sour  milk,  of  which  there  are  a  very  large  number. 
It  was  first  described  by  Hueppe  in  1884.  It  is  very  widely  dis- 
tributed. 

REFERENCES.  Hueppe,  Mitteil.  aus  dem  Kaiserl.  Gesundheits- 
amte,  1884,  Bd.  II.  p.  1837 ;  C.  149 ;  Cn.  189 ;  L.  222 ;  L.  &  N.  220. 
Mig.  2 :  327. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


b.  Agar. 


c.  Gelatin. . 


d.  Other  media. 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet.. 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

i.    MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 


5.    SPORES- 


G.    SPECIAL  CHARACTERS: — 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism . 


110  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller 's  scale)  _|_ or  — 


Gelatin  plate:    Grown  24  hours  at °C. 


(a)  Surf  ace  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C.  6  days  at 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  ACIDI-LACTICI 


111 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at 


6  days  at oC. 


Acar  Streak:    Grown  24  hours  at °€. 


48  hours  at °C. 


6  days  at °C. 


Potato :    Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Bouillon:    Grown 24  hours  at.... °C. 


48  hours  at . 


•  °C.  6  day  sat.. 


112  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATUKE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:CO2:: : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . . . 


EXERCISE  64.     SAPROGENIC  CLASS. 
Bacillus  vulgaris  (Hauser)  Mig. 


113 


SYNONYMS.     Proteus  vulgaris  Hauser.     B.  proteus  Trevisan. 

EXPLANATORY.  First  described  by  Hauser.  It  is  widely  dis- 
tributed and  is  commonly  found  in  putrefactive  substances.  It  is 
one  of  several  related  species  included  under  the  old  name  of  ' '  Bac- 
terium termo."  While  in  small  doses  and  under  ordinary  condi- 
tions it  is  harmless,  at  times,  and  in  large  doses,  it  may  be  patho- 
genic. 

REFERENCES.  Hauser,  Ueber  Faulnisbakterien,  1885 ;  C.  244 ; 
Lafar,  194-199.  Mig.  2  :  707. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE:  

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 
&.  Loeffler's  methylen-blue . 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: 

a.  Character  of  movement. . 
6.  Flagella  stain 

5.  SPORES  : 

€.    SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


114  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -|-  or  — 


Gelatin  plate:    Grown  24  hours  at °C. 


(»)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at *C. 


(a)  Surf  ace  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

!  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at 'C. 


Special  Media.:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  VULGARIS 


115 


Gelatin  Stab:    Grown  24  hours  at °C.  Q  Q 

O  O 

3          15 

£  £ 

I         I 
v .) 

48  hours  at °C.  6  days  at 

A  gar  Streak:    Grown  24  hours  at °C.  Q 

A  , 

£  £  ( 

§1 

A   I 

\ 
48  hours  at °C.  6  days  at °C. 

Potato:    Grown  24  hours  at ... o °C.  Q 

O 

1 

ii     j  i 

48  hours  at °C.  6  days  at. °C. 

Bouillon:    Grown  24  hours  at °C.  Q  Q 


48  hours  at °C. 


6  days  at °C. 


116  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C.;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.  s  PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm — closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent .- hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk . 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION . 

48  hours days 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic...  


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) ; 


SAPROGENIC  CLASS— CONTINUED. 
Bacillus  Zopfii  (Kurth)  Mig. 


117 


SYNONYMS.     Bacterium  Zopfii  Kurth;  Proteus  Zenkeri  Hauser| 

EXPLANATORY.     This  organism  belongs  to  the  group  of  putre- 
factive bacteria  ( proteus  group ) . 

REFERENCES.     Kurth,  Botan.  Zeitung,  1883 ;  C.  248 ;  Mig.  2 :  815. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  A  gar 

c.  Gelatin 

d.  Other-media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Grain's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles , 

d.  Pleomorphism 


118                                 CULTURE  CHARACTERS 
Reaction  of  media  (Fuller 's  scale)     _ ... 


or 


Gelatin  plate:    Grown  24  hours  at °C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


(a)  Surf  ace  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at *C. 


Special  Media:    (Such  as  litmus  milk  ami  blood  serum.) 


BACILLUS  ZOPFII 


119 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at oC. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at                                    °C 

Potato:     Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


120  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours per  cent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : : 

1>.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic. . . 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  65.     PHOSPHORESCENT  CLASS.  121 

Bacterium  phosphorescens  (Cohn)  Fischer. 

SYNONYMS.     Photobacterium  phosphorescens  Beijerinck. 

EXPLANATORY.  First  described  by  Fischer  in  1887.  Found  in  Kiel  harbor 
on  dead  sea  fish,  oysters  and  occasionally  on  meat  in  shops.  The  production 
of  light  is  shown  in  the  dark,  especially  when  the  organism  is  grown  on  a 
medium  made  by  boiling  two  salt  herrings  in  a  liter  of  water,  adding  100 
gms.  of  gelatin  to  the  filtrate  without  neutralization,  tubing  and  then  ster- 
ilizing (Lehmann).  Phosphorescence  can  even  be  restored  to  attenuated 
cultures  by  growth  on  this  medium.  Inasmuch  as  oxygen  is  necessary  to 
light  production  surface  growths  are  best. 

EEFERENCES.  Fisher,  Zeitschrift  fur  Hygiene,  1887,  Band  2,  p.  92;  C.  181; 
L.  &N.  231;  Mig.  2:  433. 


MORPHOLOGICAL  CHARAQTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


&.  Agrar. 


c.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

b.  Loemer's  methylen-blue . 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 
6.  Flagrella  stain 


5.    SPORES:. 


6.    SPECIAL  CHARACTERS:.... 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles.. 

d.  Pleomorphism, 


122  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -|- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at..,.,.... °C. 


6  days  at., «C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


*8  hours  at °C. 


6  days  at 0C. 


Special  Media:    (Suck  as  litmus  milk  and  blood  serum,; 


BACTERIUM  PHOSPHORESCENS 


123 


Gelatin  Stab:    Grown  24  hours  at.... ...,°C. 


48  hours  at 


'C. 


6  days  at »C. 


Agar  Streak:    Grown  24  hours  at °C. 


I 


48  hours  at 


6  days  at. . 


Potato:    Grown  24  hours  at °C. 


48  hours  at ° C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at                                  °C                r-\ 

4T 

i 

o 

Q 

CO 

1 

48  hours  at  °C.                                 6  days  at.  .  . 

C. 

124  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : . . . . 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS.... 
desiccation,  light,  disinfectants,  etc. 


4.    PIGMENT  PRODUCTION: 


5       GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

1).  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: .. 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin . ...       oCigestion  of  casein. 

diastatic , 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


126  GENERAL  BACTERIOLOGY 


CHAPTER  V 
BACTERIOLOGICAL  ANALYSIS 


EXERCISE  66.     COMPARATIVE  ANALYSIS  OF  AIR  (Kocn). 

a.  Plate  three  tubes  of  gelatin  and  expose  by  removing  lid  for 
20  minutes  in  the  following  places:  1.  Laboratory.  2.  Cellar. 
3.  Out  of  doors. 

~b.  Replace  the  lids  and  keep  plates  at  22°  C.  for  several  days. 

c.  Count  the  colonies.     The  counting  is  facilitated  by  the  use  of 
Plate  II.  on  which  the  Petri  dishes  are  to  be  placed.     In  counting 
a  hand  lens  magnifying  about  5  diameters  should  be  used.     Where 
possible  all  of  the  colonies  on  the  plate  should  be  counted,  if  this 
be  impossible  count  a  representative  area  and  estimate  the  whole 
number. 

d.  Express  the  results  in  terms  of  the  number  of  organisms 
which  fall  per  square  foot  per  minute.     The  area  of  the  Petri  dish 
can  be  read  off  directly  from  Plate  II.  in  square  centimeters,  or  it 
can  be  calculated  by  multiplying  the  square  of  the  diameter  by 
0.785. 

This  method  enables  one  to  make  a  rough  comparison  of  the 
number  of  organisms  occurring  in  the  localities  examined,  but  to 
determine  the  number  per  volume  the  following  method  must  be 
employed. 

REFERENCE.     H.  477. 

EXERCISE  67.     QUANTITATIVE  DETERMINATION  OF  NUMBER  OF 
BACTERIA  IN  AIR  (PETRI-SEDGWICK). 

GENERAL  DIRECTIONS. 

a.  A  piece  of  glass  tubing  6  mm.  (14  in.)  in  diameter  by  15  cm. 
(6  in.)  long  is  drawn  out  at  one  end  in  a  gas  flame  and  sealed. 

ft.  Fill  this  tube  about  one-third  full  with  granulated  sugar, 
insert  a  cotton  plug  next  to  the  sugar,  and  one  at  the  end  of  the  tube 
(Fig.  25,  A).  ' 


128 


GENERAL  BACTERIOLOGY 


hours  at  130' 


c.  Sterilize  in  the  hot  air  sterilizer  for  1  and 
C.  (sugar  melts  at  a  higher 
temperature). 

d.  Fasten     the     tube, 
pointed  end  up,  in  a  clamp, 
remove     the     first     cotton 
plug  and  connect  with  an 
aspirator  (Fig.  26). 

e.  Break  off  the  pointed 
end  of  the  tube  and  draw  a 
measured   quantity  of   air 
through  the  sugar. 

SPECIAL  DIRECTIONS. 
a.  Filter    50    liters    of 

air-  FIG.  26.    Aspirator  for 

ft.    Dissolve  SUgar  in   10  filtering  air. 

cc.  of  sterile  water   (water-blank)   and  make 
FIG.  25.  Apparatus  f or fli-  plates,  using  1  cc.  of  the  mixture. 

tering  air  through  sugar.   r  , 

A,  ready  for  sterilization;         c.  Incubate,  count  colonies  as  above  and  es- 

B,  point  broken  off  and  at-  .  . 

tached  to  aspirator;  a  and  timate  the  number  of  organisms  per  liter  or  air. 

6,  cotton  plugs;    c,  sugar; 

d,  clamp;  e,  rubber  tube.  ,     __ 

KEFERENCES.     A.  604;  H.  477;  L.  &  K. 
392 ;  M.  &  R.  123 ;  McF.  230. 


EXERCISE  68.     WATER  ANALYSIS. 

COLLECTION.  Water  for  analysis  must  be 
collected  in  a  sterile  vessel.  A  test-tube  or  flask 
may  be  used  in  the  laboratory,  but  when  the 
collection  is  made  outside  a  sterile  glass-stop- 
pered bottle  should  be  used.  In  collecting, 
special  care  should  be  taken  to  get  a  fair  sample ; 
if  the  water  be  in  a  reservoir,  or  the  like,  the 
bottle  should  be  filled  below  the  surface  to  avoid 
the  scum  and  away  from  the  bottom  to  avoid  the 
sediment.  Fig.  27  shows  a  form  of  apparatus 
used  to  take  samples  in  deep  water.  If  some 
time  must  necessarily  elapse  between  the  collec- 
tion of  the  sample  and  its  examination  it  should 
be  packed  in  ice.  Specially  constructed  shipping 
cases  are  used  in  most  laboratories. 


o 


FIG.  27.  Russell's 
Water  Sampler,  a, 
test-tube  from  which 
the  air  has  been  ex- 
hausted; &,  glass  tube, 
sealed;  c,  sinker;  d, 
weight  to  be  dropped 
at  proper  depth. 


130  GENERAL  BACTERIOLOGY 

QUANTITATIVE  ANALYSIS. 

a.  After  shaking  the  sample  at  least  25  times  remove  1  cc.  of 
the  water  by  means  of  a  sterile  pipette  and  place  it  in  the  bottom  of  a 
sterile  Petri  dish.  In  the  same  way  remove  %  cc.  and  TV  cc.  Pipettes 
graduated  to  TVths.  may  be  used,  or  a  1  cc.,  or  even  a  5  cc.  pipette 
may  be  used  by  counting  the  whole  number  of  drops  delivered  and 
then  taking  the  number  of  drops  to  make  the  required  fraction.  If 
the  sample  be  supposed  to  be  highly  infected  it  should  be  diluted 
with  sterile  water  before  the  cultures  are  made.  Plates  ought  not 
to  contain  over  200  colonies. 

&.  Pour  into  the  dishes  fluid  gelatin  (not  warmer  than  42°  C.) 
and  tip  them  from  side  to  side  until  the  medium  and  water  are 
thoroughly  mixed.  Solidify  and  incubate  at  22°  C.,  or  below. 

c.  In  the  same  way  make  agar  plates  using  ordinary  agar  or, 
better,  5%  glycerine  asrar.     Incubate  at  22°  C. 

d.  Count  the  colonies  at  the  end  of  48  hours  as  directed  above 
(66c.)  and  at  intervals  afterward  until  the  maximum  number  of 
colonies  is  obtained.     Express  the  results  in  the  number  of  bacteria 
per  cc.  of  watei*. 

QUALITATIVE  ANALYSIS. 

a.  Number  of  species.  Examine  carefully,  under  the  low  power 
of  the  microscope,  the  plates  made  above  to  determine  the  number 
of  different  species,  describing  each  very  briefly.  Estimate  also  the 
total  number  of  liquefying  organisms  per  cc. 

&.  Tests  for  Fecal  Bacteria  (B.  coli)  as  follows: 

1.  Fermentation  tube  test.     Inoculate  three  fermentation  tubes, 
containing  1%  dextrose  bouillon,  with  -j^,  1,  and  10  cc.  of  water 
and  incubate  at  38°  C.     Tubes  which  develop  from  30  to  70%  of 
gas  should  have  lactose  litmus  agar  plate  cultures  made  from  them 
and  then  the  gas  formula  may  be  determined.     For  B.  coli  it  will 
be  about :  C02  :  H  : :  1  :  2  or  CO,  _    ^ 

H      =Y 

2.  Indol  test.     Tubes  of  sugar-free  bouillon  or  Dunham's  solu- 
tion inoculated  and  incubated  at  38°  C.  for  4  to  5  days  will  show 
the  presence  of  indol  if  B.  coli  be  present. 

3.  Acid  colonies.     A  lactose  litmus  agar  plate  should  be  made 


132 


GENERAL  BACTERIOLOGY 


(using  about  1  cc.  of  water)  also  one  from  fermentation  tube  and 
kept  at  38°  C.     Examine  24  hours  later  for  acid  colonies. 
c  .  Pathogenic  Bacteria.     See  Chapter  X. 

REFERENCES.  A.  579  ;  H.  457  ;  McF.  234  ;  M.  &  R.  133  ;  P.  245  ; 
Prescott  &  Winslow,  Elements  of  Water  Bacteriology.  For  the 
determination  of  the  various  species  present  see  Frankland's  Micro- 
organisms of  Water  ;  Fuller  :  Keport  Am.  Public  Health  Assoc.,  1899, 
580;  Chester. 

SPECIAL  DIRECTIONS.  Analyze  a  surface  water  (lake  or  river) 
and  a  deep  well  or  a  spring  water. 


EXERCISE  69.     ESTIMATION  OF  NUMBER  OF  BACTERIA  IN  SOIL. 

a.  With  a  sterile  knife  collect  a  sample  of  soil  in.  a 
sterile  test-tube  or  Petri  dish.  Samples  at  various 
depths  can  be  secured  by  means  of  an  earth  borer. 
(Fig.  28.) 

&.  Weigh  out  1  gram  and  dilute  1000  times  with 
sterile  water. 

c.  Make  three  gelatin  plate  cultures  using  1  cc., 
%  cc.  and  TV  cc.  of  this  suspension.     Incubate. 

d.  Count  the  colonies  as  they  develop  and  estimate 
the  number  of  bacteria  per  gram  of  soil. 

e.  Many  of  the  bacteria  of  the  soil  are  anaerobic 
and  can  only  be  grown  in  the  absence  of  free  oxygen. 
See  Part  II.  Chap  VII.  for  methods  of  cultivation. 

REFERENCES.     A.  609  ;  H.  481  ;  M.  &  R.  128  ;  McF.  FIG.  28. 
240  ;  Cn.  3.  e1'8  Soii 


EXERCISE  70.     QUANTITATIVE  ANALYSIS  OF  MILK. 

a.  Obtain  a  sample  of  milk  in  a  sterile  vessel. 
6.  Dilute  milk  1000  times  with  sterile  water. 

c.  Make  plates  as  under  soil  (69). 

d.  Count  colonies  and  estimate  number  of  bacteria  per  cc. 


EXERCISE  71.     EFFICIENCY  OF  PASTEURIZATION. 

a.  Place  same  milk  as  used  in  previous  experiment  in  the  bot- 
tles of  a  pasteurizing  apparatus,  such  as  Freeman's,  and  pasteurize 
as  per  printed  directions.  Or,  place  the  milk  in  ordinary  milk  bot- 


134 


GENERAL  BACTERIOLOGY 


>V-/=J 


ties  or  fruit  jars,  filling  to  a  uniform  level;  these  are  then  to  be 

placed  in  a  flat  bottomed  pail 

(Fig.  29)  which  is  to  be  filled 

with  water  and  heated  to  71° 

C.  (160°  F.).     Remove  source 

of  heat,  cover  and  allow  tc 

stand    30    minutes.     Remove 

bottles  and  cool  as  quickly  as 

possible    without    danger    to 

glass.  FIG.  29.    Home-Made  Pasteurizers. 

~b.  Determine  bacterial  content  of  pasteurized  product  by  mak- 
ing plates.  A  dilution  of  100  will  probably  be  sufficient.  Express 
results  so  as  to  indicate  per  cent,  of  organisms  destroyed  by  the 
process.  Compare  the  keeping  qualities  of  the  pasteurized  pro- 
duct with  that  of  the  raw  milk  by 
keeping  samples  of  both  under  similar 
conditions,  e.  g.  in  locker  or  ice  chest, 
making  frequent  observations. 

Pasteurized  milk  should  not  have 
a  permanently  cooked  taste. 

REFERENCES.  H.  485;  Wis.  Exp, 
Station  Bull.  No.  44  and  18th.  An. 
Rept.  185.  Russell,  Outlines  of  Dairy 

FIG.  30.    The  Freeman  Pasteurizer.      Bacteriology,    (5th  Edit.)    113. 
EXERCISE  72.     TESTING  ANTISEPTIC  ACTION  OF  CHEMICALS. 

GENERAL  DIRECTIONS. 

a.  Fill  a  number  of  test-tubes  with  a  measured  quantity  of  agar 
(5  ce). 

&.  Add  to  the  agar  varying  but  measured  amounts  of  the  sub- 
stance to  be  tested.  If  the  antiseptic  be  not  volatile,  or  affected  by 
heat,  sterilize. 

c.  Inoculate  the  tubes  thus  prepared,  together  with  a  control, 
with  B.  coli  or  M.  pyogenes  and  make  rolls. 

d.  Keep  these  cultures  under  observation  in  the  incubator. 

e.  If  no  growth  appears  within  96  hours  repeat  the  experiment, 
using  smaller  amounts  of  the  antiseptic.     In  this  way  determine  the 
amount  of  chemical  (in  %)  which  just  prevents  growth. 

SPECIAL  DIRECTIONS.  Test  in  this  way  carbolic  acid  (5%), 
alcohol  (95  %). 

REFERENCES.     A.  619 ;  H.  506 ;  M.  &  R.  140 ;  McF.  248. 


136  GENERAL  BACTERIOLOGY 

EXERCISE  73.     TESTING  DISINFECTING  ACTION  OF  CHEMICALS. 

SUSPENSION   METHOD. 

a.  Make  a  culture  of  the  organism  to  be  studied  in  tubes  of 
bouillon  containing  5  cc. 

&.  Incubate  at  38°  C.  for  24  hours. 

c.  Add  to  this  an  equal  amount  (5  cc.)  of  the  disinfectant  to  be 
tested,  of  double  the  required  strength. 

d.  At  the  end  of  5,  10,  20,  40  and  60  minutes  make  agar  rolls, 
using  two  or  three  loopfuls  of  the  mixture  for  each  roll. 

e.  In  this  way  determine  the  time  of  exposure  necessary  to  kill 
the  organism  used. 

/.  Test  in  this  way  the  value  of  corrosive  sublimate  (1:1000) 
and  Lysol  (5%),  using  B.  coli  or  M.  pyogenes.vsx.  aureus. 

COVER-GLASS  METHOD. 

a.  Make  a  bouillon  culture  of  the  organism  to  be  studied  and 
incubate  at  38°  C.  for  24  hours. 

6.  By  means  of  a  burette,  pipette,  or  loop,  place  the  same  sized 
drop  on  each  of  several  sterile  cover-glasses  and  dry  as  directed  in 
the  experiment  on  desiccation  (43). 

c.  When  the  cover-glasses  are  dry,  they  are  to  be  immersed  in 
the  disinfectant   for  the  stated  periods   of  time;   then   removed, 
washed  in  sterile  water  and  transferred  to  tubes  of  melted  agar 
which  are  then  made  into  rolls. 

d.  Test  by  this  method  carbolic  acid  (5%),  alcohol  (95%)  and 
formaldehyde  (4%)  or  formalin  (10%),  using  B.  coli. 

REFERENCES.     A.  611 ;  McF.  249  ;  N.  518 ;  P.  152 :  S.  158. 


PLATE  I 


ooooooooo       o 
»-<»  «n<o'**ww       ° 


Facing  page  136>, 


O  O  O  O  O 

to  ^  in  ©  r*- 

GASOMETER  FOB  FERMENTATION  TUBE. 

(See  page  72). 


PLATE  II 


100 


a. 


b. 


PLATE  COUNTER  (Modified  from  Jeffers). 
FOR  COUNTING  COLONIES  OF  BACTERIA. 


The  cross  lines  divide  the  figure  into  square  centimeters.  The 
numbers  indicate  the  area  of  the  various  discs.  The  area  of  each 
sector  (a.  and  b.)  is  one-tenth  of  the  whole  area.  (See  page  126.) 

Pacing  page  137. 


PART  II 
MEDICAL  BACTERIOLOGY 


138  MEDICAL  BACTERIOLOGY 


PART  II-MEDICAL  BACTERIOLOGY 
CHAPTER  VI 

PATHOGENIC  AEROBES 


EXERCISE  74.     PREPARATION  OF  CULTURE  MEDIA. 

The  following  media  will  be  necessary  for  the  work  outlined  in 
the  following  chapters.     This  is  exclusive  of  a  few  special  media 
which  are  described  under  special  heads  and  are  to  be  made  as  a 
part  of  the  exercises  in  which  they  are  used. 
100  tubes  of  agar. 

12  tubes  of  dextrose  agar. 
100  tubes  of  gelatin. 

12  tubes  of  dextrose  gelatin. 

30  tubes  of  bouillon. 

10  fermentation  tubes  of  dextrose  bouillon. 

35  tubes  of  potato. 

35  tubes  of  litmus  milk. 

35  tubes  of  dextrose  free  broth  or  Dunham's  solution. 

30  water  blanks. 

30  tubes  of  Loeffler  's  blood  serum.  This  is  prepared  as  follows : 
a.  Collection  of  the  blood.  Sterilize  Mason  fruit  jars,  by  suc- 
cessive washings  in  corrosive  sublimate,  distilled  water,  alcohol  and 
ether  (or  a  large  pail  may  be  used).  These  are  to  be  carried  to 
the  slaughter  house  and  the  blood  from  a  beef  caught  directly  into 
them.  The  blood  is  then  allowed  to  stand  undisturbed  for  15  to 
30  minutes,  or  until  the  clot  has  firmly  attached  itself  to  the  sides 
of  the  vessels,  when  they  are  to  be  covered  and  removed  to  the 
laboratory. 

6.  Separation  of  the  serum  from  the  blood  clot.  The  clot  is 
separated  from  the  sides  of  the  vessel  by  means  of  a  sterile  knife  or 
glass  rod,  and  the  vessel  placed  in  the  ice  chest.  After  standing 
48  hours  the  clot  will  have  shrunken  away  from  the  walls  of  the 


140 


MEDICAL  BACTERIOLOGY 


vessel  leaving  the  clear  serum  on  the  top  and  at  the  sides.  This 
can  now  be  pipetted  or  siphoned  off.  If  the  serum  contains  a  large 
number  of  red  blood  corpuscles  it  can  be  placed  in  rather  tall  cylin- 
ders (graduates)  and  allowed  to  stand  24  hours  longer,  when  the 
clear  straw  colored  serum  can  be  readily  separated.  This  may  be 
preserved  for  a  long  time  by  the  addition  of  %%  chloroform  and 
kept  in  a  tightly  corked  bottle  in  a  cool  place. 

c.  Loeffler's  mixture.     This  consists  of  3  parts  of  blood  serum 
and  1  part  of  \%  dextrose  bouillon. 

d.  Sterilization.     Fill  sterile  test-tubes  (about  3  cm.  deep)  with 
the  serum  mixture  and  place  them  im- 

mediately in  a  sloping  position  in  an 
inspissator  (Fig.  31),  or  steamer  and 
heat  to  95°  C.  for  1  hour  on  three  con- 
secutive days.  If  a  higher  tempera- 
ture be  employed  bubbles  are  formed 
which  rupture  the  surface  of  the  me- 
dium in  their  escape.  When  steril- 
ized the  tubes  should  be  sealed  with 
paraffin  or  otherwise. 

REFERENCES.     A.  110;  H.  51;  M. 


&T?    AQ     i\/r«Tn    1  on     XT 
K.  4o  ;  Me.b  .  Io7  ;  JN. 


T> 
r. 


PIG.  31.    Blood  Serum  Inspissator, 
(Muir  &  Ritchie). 


EXERCISE  75.     ERYSIPELAS  GROUP. 
Streptococcus  erysipelatos  FEHLEISEN. 


HI 


SYNONYMS.     Streptococcus  pyogenes  ROSENBACH  ;  streptococcus. 

EXPLANATORY.  First  described  by  Fehleisen.  It  is  found  in 
abscesses,  pyemia,  puerperal  fever  and  erysipelas.  It  is  frequently 
present  in  mixed  or  secondary  infections,  and  occurs  in  the  mouth 
and  sputum  and  on  the  mucous  membranes  of  the  nose,  urethra, 
vagina,  etc. 

REFERENCES.  Fehleisen,  Aetiol.  des  Erysipels,  Berlin  1883 ; 
A.  279 ;  C.  65 ;  H.  165 ;  K.  &  W.  Ill,  303 ;  L.  &  N.  135 ;  Mig.  2,  6 ; 
M.  &  R.  184 ;  McF.  262 ;  P.  476. 


MORPHOLOGICAL.  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

?> .  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE  : 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet 

6.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement 

6.  Flagrella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: , 

a.  Capsules , 

6.  Involution  forms , 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


142  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  4. or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  :  (b)  Deep  Colonies. 


Sketches. 


op 

•c 

Special  Media:    (Sucb  as  liinaus  milk  and  blood  serum.) 


STREPTOCOCCUS  ERYSIPELATOS 


143 


Gelatin  Stab:    Grown  24  hours  at  .                       °C               *••} 

Q 

0 

i 

•8 

£ 

2 

§ 

§ 

J3 

V    ) 

A 

V     ) 

48  hours  at °C. 


6  days  at C. 


Agar  Streak:    Grown  24  hours  at 


48  hours  at 


days  at °C. 


Potato:    Grown  24  hours  at 


48  hours  at .   °C. 


6  days  at.. 


Bouillon:    Grown  24  hours  at           ..  .                   °C                r^ 

ts 

oo 

O 

v  2 

48  hours  at »C. 


6  days  at °C. 


144  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE  : 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes ; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION; 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture : 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic...  


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  76.     PUS  COCCUS  GROUP. 
Micrococcus  pyogenes  var.  albus  (EOSENBACH)  L.  &  N. 


145 


SYNONYMS.  Staphylococcits  pyogenes  albus  KOSENBACH; 
Staphylococcus  epidermidis  albus  WELCH  ;  white  staphylococcus. 

EXPLANATORY.  First  described  by  Rosenbach.  One  of  the  com- 
mon organisms  found  in  pus.  Occurs  on  the  skin,  in  sputum,  air, 
water,  dust  and  soil. 

REFERENCES.  Rosenbach,  Mikroorganismen  bei  dem  Wundinfec- 
tionskrankheiten  des  Menschen.  1884;  C.  75 ;  K.  &  W.  Ill,  105; 
L.  &  N.  180 ;  Mig.  2,  87 ;  McF.  255  :  P.  470. 


MORPHOLOGICAL  CHARACTERS 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

f>.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


146  CULTURE  CHARACTERS 

Eeaction  of  media  (Puller's  scale)  -|_   


or  — 


Gelatin  plate:    Grown  24  hours  at °C. 


a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


MICROCOCCUS  PYOGENES  var.  ALBUS 


147 


Gelatin  Stab:    Grown  24  iiours  at*.  ..........        °C                 r^ 

0 

? 

« 

1 

O 

\             / 

"S 

\    / 

48  hours  at 


'C. 


6  days  at 0C. 


Agar  Streak:    Grown  24  hours  at , 


48  hours  at °C. 


Gdaysat °C. 


Potato :     Grown  24  hours  at °C. 


48  hours  at  °C. 

6  days  at                             •         °C 

Bouillon:    Grown  24  hours  at. 


Q 


48  hours  at °C. 


6  days  at °C. 


148  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C.;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes ; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3     RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

'gas  formula,  H:  COz:  : : 

&.  lactose c.  saccharose 

6.     ACID  OR  ALKALI    PRODUCTION : 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . , 


PUS  COCCUS  GROUP— CONTINUED. 


149 


Micrococcus  pyogenes  var.  aureus  (KOSENBACH)  L.  &  N. 

SYNONYMS.  Staphylococcus  pyogenes  aureus  ROSENBACH  ;  Golden 
pus  coccus. 

EXPLANATORY.  First  described  in  1884  by  Rosenbach.  It  is  the 
most  common  organism  in  pus— 80%. 

REFERENCES.  Rosenbach,  Mikroorganismen  bei  dem  Wundin- 
fectionskrankheiten  des  Menschen,  1884;  A.  270;  C.  89;  Fl.  2,  96; 
H.  162;  K.  &  W.  III.  105;  L.  &  N.  180;  Mig.  2,  135;  M.  &  R.  182; 
McF.  256  ;  P.  461. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

«.  Character  of  movement. . 
6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


150  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -}_   or  — 


Gelatin  plate:    Grown  24  hours  at ,...°C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches 


48  hours  at. 


6  days  at.,...,... .»C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches, 


48hoursat. 


6  days  at. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


MICROCOCCUS  PYOGENES  var.  AUREUS 


151 


Gelatin  Stab :    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at oC. 


Agar  Streak:    Grown  24  hours  at °C. 


hours  at °C. 


6daysat °C 


Potato:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at ....°C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at. °C 


152 


PHYSIOLOGICAL  CHARACTERS 


lo    RELATION  TO  TEMPERATURE: 

optimum. °C. ;  limits to °C.: 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: '. 

3.  RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— 

4.  PIGMENT  PRODUCTION  : 

5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm . 

rate  of  development:  24  hours per  cent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

l».  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 

litmus  milk 

7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia 

8.  INDOL  PRODUCTION 

48  hours days 

9.  ENZYME  PRODUCTION: 

proteoly  tic 

digestion  of  gelatin digestion  of  casein 

diastatic • 

10.  CHARACTERISTIC  ODOK: 

11.  PATHOGENESIS  (or  other  special  characters):. 


EXERCISE  77.     MALTA  FEVER  GROUP.  153 

Micrococcus  melitensis  BRUCE. 

EXPLANATORY.  This  organism  is  the  cause  of  Malta  fever  and 
is  found  especially  in  the  spleen  of  the  diseased. 

REFERENCES.  Bruce,  Practitioner,  Sept.  1887  and  Ann.  de 
1'Inst.  Pasteur,  1893,  7,  289;  Durham,  Jour.  Path,  and  Bact.,  1898, 
5,  377  ;  H.  441 ;  K.  &  W.  Ill,  438 ;  L.  &  N.  168 ;  Mig.  2,  83  ;  McF.  581 ; 
M.  &  R.  452. 


MORPHOLOGICAL  CHARACTERS: 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


I.  Agar 


c.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 


5.    SPORES: 


&     SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms , 

c.  Deposits  or  vacuoles.. 

d.  Pleomorphism 


154  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  _j_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies,  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at "C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C.  6  days  at 


Special  Media:    (Such  as  litmus  milk  and  blood  seruinj 


MICROCOCCUS  MELITENSIS 


155 


Gelatin  Stab:    Grown24hours  at °C. 


48  hours  at . . 


6  days  at oC. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at. 


Potato :    Grown  24  hours  at °C. 


O 


48  hours  at 


6  days  at °C. 


Bouillon:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


156  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

«.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours per  cent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2-.  : : 

?>.  lactose c.  saccharose 

6.  ACID  OR  ALKALI    PRODUCTION : -  - » , , 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites .  - to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . , 


EXERCISE  78.     DIPLOCOCCUS  GROUP.  157 

Micrococcus  gonorrhoeae  (BAUM)  FLUEGGE. 

SYNONYMS.     Gonococcus;  Diplococcus  gonorrhoeae  BAUM. 

EXPLANATORY.  First  described  in  1879  by  Neisser.  It  is  con- 
stantly found  in  gonorrhoeal  discharges  and  may  produce  disease 
on  any  mucous  membrane;  urethra,  bladder,  rectum,  conjunctiva 
(causing  ophthalmia  neonatorum),  and  even  cause  arthritis  (gonor- 
rhoeal rheumatism),  endocarditis,  salpingitis  and  general  septicemia. 

REFERENCES.  Neisser,  Cent.  f.  d.  Mediz.  Wissensch.,  1879,  497; 
Foulerton,  Trans.  Brit.  Inst.  of  Prev.  Med.,  1897,  1,  40 ;  A.  288 ; 
C.  72 ;  H.  179  ;  K.  &  W.  Ill,  148 ;  L.  &  N.  164 ;  Mig.  2,  188 ;  M.  &  R. 
189  ;  McF.  275  ;  P.  522. 

CULTURE  CHARACTERS. 


MORPHOLOGICAL  CHARACTERS: 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media, > 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain » 

d.  Special  stains, 

4.  MOTILITY: 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL,  CHARACTERS: 

a.  Capsules 

b.  Involution  forms «... 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


158  CULTURE  CHARACTERS 

The  Micrococcus  gonorrhoeae  does  not  grow  on  the  ordinary  arti- 
ficial media  but  may  be  cultivated  on  the  following : 

a.  Blood  agar.  Blood  drawn  from  the  finger,  under  aseptic  pre- 
cautions, into  a  capillary  pipette  is  placed  on  the  surface  of  agar 
either  in  tube  or  Petri  dish.  This  blood  is  then  inoculated  with 
the  material  containing  the  organism  (pus  or  pure  culture)  and 
smeared  over  the  surface  of  the  agar  either  with  the  loop,  or  better, 
by  means  of  a  sterile  camel's  hair  brush. 

&.  Wertheim's  method.  Human  blood-serum  (from  placenta  or 
pleuritic  or  other  effusion  may  be  used)  in  a  fluid  and  sterile  condi- 
tion is  placed  in  two  or  three  test-tubes.  These  are  heated  to  40°  C. 
and  inoculated  with  the  material  containing  the  organism,  making 
dilutions  from  one  to  another,  if  necessary.  To  each  tube  is  then 
added  an  equal  quantity  of  nutrient  (ordinary  or  2%)  agar  thor- 
oughly liquefied  and  cooled  to  40°  C.  The  two  are  then  thoroughly 
mixed  and  quickly  poured  into  Petri  dishes  and  placed  in  the  incu- 
bator at  38°  C.  Colonies  appear  in  24  hours. 

c.  Eabbit  blood-serum  may  be  used  either  in  a  fluid  or  solid 
condition. 


DIPLOCOCCUS  GROUP— CONTINUED. 


159 


Micrococcus  Weichselbaumii  (TREVISAN). 

SYNONYM.  Diplococcus  intracellularis  meningitidis  WEICHSEL- 
BAUM. 

EXPLANATORY.  First  described  in  1887  by  Weichselbaum.  It  is 
found  in  the  meningeal  exudate  of  certain  cases  of  epidemic  cerebro- 
spinal  meningitis  and  in  nasal  secretions  in  a  number  of  cases. 

REFERENCES.  Weichselbaum,  Fortschritte  der  Medicin,  1887 ; 
Councilman,  Kept.  Mass.  State  B.  of  H.  1898  ;  A.  298  ;  C.  64 ;  H.  170  ; 
K.  &  W.  Ill,  256 ;  L.  &  N.  148  ;  Mig.  2,  189  ;  McF.  281 ;  M.  &  R.  188 ; 
P.  516;  S.  310. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media. 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet 

&.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT  Y  : 

a.  Character  of  movement — 

&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS : 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


160  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  _}_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies 


Sketches, 


48  hours  at. °C, 


6  days  at. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


48  hours  at. 


6  days  at. 


Sketches. 


Special  Media:    (Such  as  litmus  inilk  and  blood  serum.) 


MICROCOCCUS  WEICHSELBAUMII 


161 


Gelatin  Stab:    Grown  24  hours  at °C. 


•i 


48  hours  at , 


6  days  at oC. 


Agar  Streak:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 


48  hours  at »C. 


6  days  at °<X 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at... °C. 


162  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: , 

optimum °C.;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 



4.    PIGMENT  PRODUCTION: 


6.     GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

6.  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.    REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

80  INDOL  PRODUCTION 

48  hours days. 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic...  


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  79.     SARCINA  GROUP. 


163 


Sarcina  tetragena  (GAFF  KY)  MIG. 
SYNONYM.     Micrococcus  tetragenus  GAFFKY. 

EXPLANATORY.  First  described  in  1883  by  Gaffky.  It  is  found 
in  phthisical  eavities  and  in  sputum,  and  it  occasionally  occurs  in 
mixed  infections  as  abscesses  connected  with  carious  teeth,  about  the 
neck,  jaws,  and  middle  ear ;  rarely  elsewhere. 

REFERENCES.  Gaffky,  Langenbeck's  Archiv,  1883,  28,  500;  A. 
326 ;  C.  84 ;  Fl.  2, 155  ;  H.  172 ;  L.  &  N.  171 ;  Mig.  2,  225 ;  M.  &  R.  187  ; 
M.  &  W.  133 ;  McF.  571 ;  P.  472. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

•* 

b.  A  gar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles , 

d.  Pleomorphism 


164  CULTURE  CHARACTERS 

Keaction  of  media  (Fuller's  scale)  -J-   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf ac*  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at.  *C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


SARCINA  TETRAGENA 


165 


Gelatin  Stab:    Grown  24  hours  at. 


48  hours  at °C. 


6  days  at oC. 


Agar  Streak:     Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Potato:     Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


166  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: , 


3-    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: c 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic , 

digestion  of  gelatin digestion  of  casein . 

diastatic... 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  80.     ANTHRAX  GROUP. 


167 


Bacterium  anthracis  (KOCH)  MIG. 

SYNONYMS.     Bacillus  anthracis  KOCH  ;  anthrax  bacillus. 

EXPLANATORY.  First  described  by  Robert  Koch  in  1876.  Found 
in  the  blood  and  tissue  in  cases  of  anthrax  or  splenic  fever. 

REFERENCES.  Koch,  Cohn  's  Beitraege  zur  Biologic  der  Pflanzen, 
1876,  2,  277;  Chester,  Rept.  Delaware  Exp.  Station,  July,  1895; 
A.  492 ;  C.  190 ;  Fl.  2,  217 ;  H.  184 ;  K.  &  W.  II,  1 ;  L.  &  K.  287 ;  L.  & 
N.  307 ;  Mig.  2,  280 ;  M.  &  R.  300 ;  M.  &  W.  156 ;  McF.  469 ;  P.  547 ; 
S.  328. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin • 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

r,.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of!  movement.. 

b.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


168  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -|_ or  — 


Gelatin  plate:    Grown  24  hours  at °C.       |     Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies,  j 


48  hours  at. 


«days  at 


{a)  Surf  ace  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

(b)  Deep  Colonies. 


Sketches. 


48  hours  at                  • 

°C 

6  days  at  .  c  °C 

i 

i 

i 

Special  Media:    (Such  as  litmus  milk  and  olood  serum.) 


BACTERIUM  ANTHRACIS 


169 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at CC. 


6  days  at «,C. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Potato:     Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Bouillon:     Grown  24  hours  at °C. 


48  hours  at «C. 


6  days  at °C. 


170  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: ..4, 

optimum ° C. ;  limits to ° C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: , 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic . 


rO.    CHARACTERISTIC  ODOR: 

11.    PATHOGENESIS  (or  other  special  characters):. 


EXERCISE  81.     FRIEDLANDEE  GROUP. 


171 


Bacterium  pneumonicum  (FRIED.)  MIG. 

SYNONYMS.  Friedlander's  bacillus;  bacillus  pneumonias  WEICH- 
SELBAUM  ;  Pneumobacillus  of  Friedlander. 

EXPLANATORY.  First  described  by  Friedlander  in  1882.  Found 
frequently  in  normal  saliva,  lungs,  "rusty  sputum "  of  pneumonia, 
and  has  been  found  in  air  and  water. 

REFERENCES.  Friedlander,  Virchow's  Archiv,  32,  319;  C.  131; 
Fl.  2,  342 ;  H.  314 ;  K.  &  W.  Ill,  189 ;  L.  &  N.  225 ;  Mig.  2,  350 ;  M.  & 
R.  209  ;  McF.  300 ;  P.  458. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agrar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet.. 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILJTY: 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


172  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  4.   or  —   . 

Gelatin  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48  hours  at °C.  6  days  at °C. 

Agar  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48  hours  at °C.  6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  PNEUMONICUM 


173 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at »C. 


Agar  Streak:    Grown  24  hours  at °C. 


I    6 


48  hours  at °C. 


6  days  at °C. 


Potato:     Grown  24  hours  at 


9  \ 


48  hours  at °C. 


Sdaysat °C. 


Bouillon:     Grown  24  hours  at. 


48  hours  at °C. 


6  days  at °C. 


174  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes ; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc-:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development :  24  hours per  cent. .  48  hours per  cent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COa:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION:...  


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


FRIEDLANDER  GROUP— CONTINUED. 


175 


Bacterium  aerogenes  (ESCH.)  Mm. 

SYNONYMS.  Bacterium  lactis  aerogenes  ESCHERICH;  Bacillus 
aerogenes  KRUSE. 

EXPLANATORY.  This  organism  was  first  described  by  Escherich, 
who  isolated  it  from  the  milk-stools  of  infants.  It  is  very  similar  to 
Bact.  acidi-lactici,  and  often  difficult  to  differentiate  from  B.  coli. 
Found  in  milk,  feces,  air,  water,  etc. 

REFERENCES.  Escherich,  Fortschritte  der  Medizin,  1885,  No.  16- 
17.  C.  128 ;  L.  &  N.  221 ;  Mig.  2,  396. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other-media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL,  CHARACTERS  : 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism ,. 


176  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -j-   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

*a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at 


°C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at  

°c 

6  days  at  

°c 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  AEROGENES 


177 


Gelatin  Stab*    Grown  24  hours  at  °C               r^ 

i 

s 

O 

hours  at  °C 

15  hours  at  .  .                           .  ,  °C.                                  6  days  at  .  . 

Agar  Streak:    Grown  24  hours  at C. 


O 


O  i 


48  hours  at 


Gdaysat... °C. 


Potato :    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at.. 


Bouillon:    Grown  24  hours  at. 


dours  at 


'C. 


6  days  at. . 


178  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : , 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.: — . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H :  COa:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI    PRODUCTION: 


litmus  milk . 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION — 

48  hours days , 

9.  ENZYME  PRODUCTION: 


proteolytic - 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) ; 


FRIEDLANDER  GROUP— CONTINUED. 
Bacterium  capsulatum  (STERNBERG)  CHESTER. 


179 


SYNONYMS.     Pfeiffer's    capsule    bacillus;    Bacillus  capsulatus 
STERNBERG. 

EXPLANATORY.     First  described  by  Pfeiffer,  who  isolated  it  from 
the  blood  of  guinea  pigs  which  died  spontaneously. 

REFERENCES.     Pfeiffer,  Z.  f .  H.  1889,  6, 145  ;  C.  129  ;  L.  &  N.  228 ; 
Mig.  2,  349. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

1.  Agar r. 

c.  Gelatin 

d.  Other  media 

•• 

2.  SIZE: 

2.    STAINING  POWERS: 

a.  Aqueous  gentian- violet 

6.  Loeffler' a  tnethylen-blue 

c.  Gram's  stain 

d.  Special  stains 

&,    MOTILJT Y  : 

a.  Character  of  movement 

6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


180  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  J_   


or  — 


Gelatin  plate:     Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  :   (b)  Deep  Colonies. 


Sketches. 


48  hours  at         •  .  •      • 

°C 

°c 

\. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  CAPSULATUM 


181 


Gelatin  Stab:    Grown  24  hours  at °C.  Q 


48  hours  at 


C. 


6  days  at oC. 


Agar  Streak:     Grown  24  hours  at 


48  hours  at 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


182  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. : 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2;  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours per  cent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  €62:  : : 

6.  lactose c.  saccharose 

6.     ACID  OR  ALKALI    PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: .  . 


proteolytic 

digestion  of  erelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) 


EXERCISE  82.     SWINE  PLAGUE  GROUP.  183 

Bacterium  cholerae  (ZOPF)  KITT. 

SYNONYMS.  Bacillus  of  chicken  cholera;  Bacillus  of  swine 
plague;  Bacterium  cholerge-gallinarum  CROOKSHANK;  Bact.  suicida 
MIGULA  ;  Bacillus  septicemiae  hemorrhagicse  HUEPPE. 

EXPLANATORY.  First  described  by  Koch  in  1878.  Found  in 
blood,  organs  and  excreta  of  chickens  suffering  with  fowl  cholera, 
and  in  swine  suffering  from  swine  plague. 

REFERENCES.  Koch,  Wundinfectionskrankheiten,  Septikaemie 
bei  Kaninchen,  1878 ;  Smith,  Report  on  Swine  Plague,  Bureau  of 
Animal  Industry,  U.  S.  Dept.  Agri.,  1891 ;  Smith  &  Moore,  Bull.  6, 
B.  A.  I.,  1894 ;  C.  135 ;  H.  305 ;  K.  &  W.  II,  543 ;  L.  &  N.  208 ;  Mig.  2, 
364 ;  McF.  534. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


c.  Gelatin. 


d.  Other  media. 


2.    SIZE: 

•3.    STAINING  POWERS: 

a.  Aqueous  gentian-violet.. 

&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.    MOTILITY  : 

a.  Character  of  movement.. 
6.  Flagella  stain 


5.    SPORES: 


6.    SPECIAL  CHARACTERS:.... 

a.  Capsules 

6.  Involution  forms  — 
c.  Deposits  or  vacuoles. 
«"'.  Pleomorphism 


184  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  _|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


°c 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  CHOLERAE 


185 


Gelatin  Stab:    Grown  24  hours  at °C. 


v  ./ 


48  hours  at 


6  days  at 


Agar  Streak:    Growr  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Potato:     Grown  24  hours  at 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


«  ! 


4«  hours  »t 


6  days  at. 


186  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: 

optimum °C.;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes : 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.: — . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hoars. per  cent.,  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H :  CO2:  : : 

&.  lactose c,  saccharose 

6.  ACID  OR  ALKALI    PRODUCTION : - 


litmus  milk, 


7.    REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

80  INDOL  PRODUCTION 

48  hours. days . 

9.  ENZYME  PRODUCTION: .  . 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . . 


SWINE  PLAGUE  GROUP— CONTINUED. 


187 


Bacterium  bovisepticum  (KRUSE)  MIG. 

SYNONYMS.     Bacillus  of  hemorrhagic  septicemia;  Bacillus  bovi* 
septicus  KRUSE. 

EXPLANATORY.     First  described  by  Bellinger,  1878.     It  is  the 
cause  of  hemorrhagic  septicemia  in  cattle  and  in  other  animals. 

KEFERENCES.     Bellinger,   Ueber   eine  neue   Wild  und  Rinder- 
seuche,  Muenchen,  1878 ;  C.  137 ;  K.  &  W.  II,  559 ;  Mig.  2,  367. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 
&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL.  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c,  Deposits  or  vacuoles 

d.  Pleomorphism......  ..... 


188  CULTURE  CHARACTERS 

"Reaction  of  media  (Fuller's  scale)  -J_      .... 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


40  uours  at °C. 


6  days  at «C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


»C 

«c. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  BOVISEPTICUM 


189 


Gelatin  Stab:    Grown  24  hours  at. 


48  hours  at °C. 


6  days  at oC. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Potato:    Grown  24  hours  at 


48  hours  at , °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at. 


48  hours  at °C. 


6  days  at °C. 


190  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: c «,.... 

optimum °C. ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made : 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. : — . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours per  cent. 

72  hours per  cent., .hours per  cent. 

reaction  iu  open  arm 

gas  formula,  H:  COa:  : : 

fe.  lactose « c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION : -  - 


litmus  milk. 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia.. . 

8.  INDOL  PRODUCTION 

48  hours days . 

8.    ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic , 


10.  CHARACTERISTIC  ODOR:..... 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXEECISE  83.  GLANDERS  GROUP. 


191 


Bacterium  mallei  (LOEFFLER)  MIG. 
SYNONYMS.     Bacillus  mallei  LOEFFLER  ;  Bacillus  of  Glanders. 

EXPLANATORY.  First  described  by  Loeffler  in  1886.  Found  in 
the  nodules,  ulcers,  discharges,  etc.,  of  glanders  or  farcy. 

EEFERENCES.  Loeffler,  Arbeit,  aus  dem  Kaiserl.  Gesundheit- 
samte,  1886,  1,  141 :  A.  376 ;  H.  256 ;  K.  &  W.  II,  707 ;  L.  &  K.  300 ; 
L.  &  N.  384 ;  Mig.  2,  498 ;  M.  &  R.  275  ;  M.  &  W.  164 ;  McF.  359  ;  P.  508. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism , 


192  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  _|_     or  — 


Gelatin  plate:    Grown  24  hours  at. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  MALLEI 


193 


Gelatin  Stab:    Grown  24  hours  at ...°C. 


§ 

i      I 


48  hours  at 


3C. 


6  days  at 


Agar  Streak:    Grown  24  hours  at °C.  j-j 


V 


48  hours  at °C. 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 

05 

I 

48  hours  at °C.  6  days  at °C. 

Bouillon:    Grown  24  hours  at °C. 

•g 

'  j 

\ 
^ x, 

48hoursat °C.  I  6  days  at.. 


194  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: , 

optimum ° C. ;  limits to °C.  j 

thermal  death-point °C. ;  time  of  exposure minutes ; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

ft.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION:...   


litmus  milk , 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days , 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


GLANDERS  GROUP—  CONTINUED. 


195 


Bacterium  rhusiopathiae  (KITT.)  Mio. 

SYNONYMS.  Bacillus  of  swine  erysipelas;  Bacterium  erysipe- 
latus-suis  MIGULA. 

EXPLANATORY.  First  described  by  Loeffler  in  1882.  Found  in 
blood,  internal  organs,  etc.,  of  swine  infected  with  the  disease. 

REFERENCES.  Loeffler.  Arb.  aus  dem  Kaiserl.  Gesundheitsamte, 
1885,  1,  46 ;  C.  352 ;  K.  &  W.  Ill,  711 ;  L.  &  N.  300 ;  Mig.  2,  431 ; 
McF.  552. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agrar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: o „ 

a.  Aqueous  gentian-violet 

6.  Loeffler's  methylen-blne 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILJTY  : , 

a.  Character  of  movement „ 

&.  Flagella  stain 

5.  SPORES  : . 

6.  SPECIAL  CHARACTERS  :., 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


196  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -|_   or  — 


Gelatin  plate:    Growa  24  hours  at...,    ......  °C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches, 


48  hours  at »C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


OQ 

6  days  at  

•c 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  RHUSIOPATHIAE 


197 


Gelatin  Stab:    Grown  24  hours  at. 


4» 


48  hours  at 


6  days  at oC. 


Agar  5treaK:     Grown  24  hours  at 


°C. 


•a 

£ 


48  hours  at 


6  days  at.... °C. 


Potato:    Grown  24  hours  at 


48  hours  at 


6  days  at.. 


Bouillon:    Grown  24  hours  at °( 


48  hours  at °C. 


6  days  at °C. 


198  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: • 

optimum °C.;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION:. 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture : 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days 

9.  ENZYME  PRODUCTION: 


proteolytic • 

'digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :., 


EXERCISE  84.     DIPHTHERIA  GROUP.  199 

Bacterium  diphtherias  (LOEFFLER)   MIG. 

SYNONYMS.  Bacillus  diphtherias  LOEFFLER;  Klebs-Loeffler 
bacillus. 

EXPLANATORY.  First  described  in  1883  by  Klebs.  First  culti- 
vated in  1884  by  Loeffler.  Found  in  the  false  membrane*  in  cases  of 
diphtheria,  and  in  small  numbers  in  spleen,  liver,  etc.;  occasionally 
in  healthy  throats. 

REFERENCES.  Klebs,  Verhandl.  d.  Kongress  fuer  innere  Medi- 
zin,  1883?  II;  Loeffler,  Mitth.  aus  dem  Kaiserl.  Gesundheitsamte, 
1884,  2,  421 ;  A.  386 ;  C.  354 ;  Fl.  2,  460 ;  H.  194 ;  K.  &  W.  II.  759 ; 
L.  &  K.  207 ;  L.  &  N.  389 ;  Mig.  2,  499 ;  M.  &  R.  356 ;  M.  &  W.  137 ; 
McF.  410 ;  P.  229. 

MORPHOLOGICAL,  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet 

&.  Loeffler's  methyien-blue 

c.  Gram's  stain 

(/.  Special  stains 

4.  MOTILIT  Y : 

a.  Character  of  movement 

&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL.  CHARACTERS  : 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 


200  CULTURE  CHARACTERS 

Eeaetion  of  media  (Fuller's  scale)  -J-   or  — 


Gelatin  plate:    Grown  24  Nhours  at °C. 

a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  :   (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  DIPHTHERIAE 


201 


Gelatin  Stab:    Grown  24  hours  at °C.  Q 


I 


48  hours  at °C. 


6  days  at 


Agar  Streak:     Grown  24  hours  at °C. 


48  hours  at °C.  6  days  at 

Potato:    Grown  24  hours  at °C.  qj 

I  A.   'I 
\       ' 

/ 
) 
48  hours  at °C.  6  day s  at °C. 

Bouillon:    Grown  24  hours  at °C. 


48  hours  at ..°C. 


Gdaysat °C. 


203  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point , °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made. . . ; 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development :  24  hours per  cent. .  48  hours per  cent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COa:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia.. . 

8.  INDOL  PRODUCTION — 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteoly  tic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters):.. 


DIPHTHERIA  GROUP— CONTINUED. 


203 


Bacterium  pseudodiphtheriticum  (LOEFFLER)  MIG. 

SYNONYMS.  Pseudodipktheria  bacillus  of  LOEFFLER;  xerose  ba- 
cillus Of  NEISSER-KUSCHBERT. 

EXPLANATORY.  Isolated  by  Hoffman  and  others,  from  the 
healthy  mouth  and  throat;  by  Neisser-Kuschbert  and  others  from 
xerosis  and  other  affections  of  the  conjunctiva.  This  may  be  only  a 
non-virulent  variety  of  Bact.  diphtheriae. 

KEFERENCES.  A.  401 ;  C.  355 ;  H.  214 ;  K.  &  W.  II,  823 ;  L.  &  N. 
404 ;  Mig.  2,  503 ;  M.  &  R.  370. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

5 .  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles,, 

d.  Pleomorphism , 


204  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -j-   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


(a)  Surface  Colonies. 


A  gar  plate:    Grown  24  hours  at  ............  °C. 

(b)  Deep  Colonies. 


Sketches. 


°c 

°c. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  PSEUDODIPHTHERITICUM 


205 


Gelatin  Stab:    Grown  24  hours  at °C. 


Agar  Streak:    Grown  24  hours  at °C. 


!       \ 

— 


48  hours  at  °C 

6  days  at              oC 

48  hours  at °C. 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 


48  hours  at 


'C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C, 


48  hours  at . . .  ,°C. 


6  days  at., 


206  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. •, 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

&.  lactose c.  saccharose 

S.     ACID  OR  ALKALI   PRODUCTION : 


litmus  milk. 


7-    REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days , 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  85.     PNEUMONIA  GROUP. 


207 


Bacterium  pneumoniae  (WEICHSELBAUM)  MIG. 

SYNONYMS.  Fraenkel's  pneumococcus ;  diplococcus  pneumoniae 
WEICHSELBAUM  ;  Streptococcus  lanceolatus  GAMALEIA. 

EXPLANATORY.  First  described  by  Sternberg  in  1880.  Found 
in  saliva  and  in  the  nasal  secretion  of  healthy  persons— from  20  to  50 
per  cent.  Usually  present  in  "rusty  sputum"  of  pneumonia 
patients. 

REFERENCES.  Weichselbaum,  Wiener  Med.  Jahrbuecher,  1886 ; 
Welch,  Johns  Hop.  Hosp.  Bulletin,  1892,  3,  125 ;  A.  321 ;  C.  63 ; 
H.  310 ;  K.  &  W.  II,  823 ;  L.  &  N.  143 ;  Mig.  2,  347 ;  M.  &  R,  208 ; 
McF.  289  ;  P.  498. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet.. 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

A.    MOTILITY  : 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: 

a.  Capsules o 

l>.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


208  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -f-   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at. 


Agar  plate:    Grown  24  hours  at. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


US  hours  at °C. 


6  days  at °C. 


Special  Media:    (Suck  as  litmus  milk  and  blood  serum.) 


BACTERIUM  PNEUMONIAE 


209 


Gelatin  Stab:    Grown  24  hours  at °C. 


Q 


48  hours  at 


6  days  at oC. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at 


>c. 


Potato:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at.. 


210  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE  : , 

optimum °C.;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:...  ..'  . 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : :.. 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) 


EXERCISE  86.     INFLUENZA  GROUP. 


211 


Bacterium  influenzae  (PFEIFFER)  L.  &  N. 

SYNONYM.     Bacillus  influenzae  PFEIFFER. 

EXPLANATORY.  First  described  in  1892  by  R.  Pfeiffer.  Found 
in  the  sputum  and  in  nasal  secretions  of  the  diseased. 

REFERENCES.  Pfeiffer,  Z.  f .  H.  1993,  13 ;  357 ;  A.  371 ;  C.  351 ; 
Fl.  2  ;  434 ;  H.  316  ;  K.  &  W.  Ill,  359 ;  L.  &  N.  202 ;  Mig.  2,  506 ;  M.  & 
R,  430;McF.  574;  P.  320. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon , 

&.  Agar 

c.  Gelatin 

d.  Othermedia 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES  : .  o 

6.  SPECIAL,  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


212  CULTURE  CHARACTERS 

B.  influenzae  does  not  grow  on  the  ordinary  artificial  culture 
media,  but  may  be  cultivated  on  agar  slopes  upon  the  surfaces  of 
which  blood  has  been  smeared.  The  blood  from  man,  rabbits, 
guinea-pigs  and  frogs  can  be  used,  but  that  from  pigeons  is  best. 
The  blood  may  be  obtained  from  a  needle  prick  and  spread  over 
the  medium  with  a  loop.  The  skin  should  first  be  washed  with 
alcohol  and  then  ether  and  the  first  drops  should  not  be  used.  The 
sterility  of  these  tubes  should  be  tested  by  placing  them  in  an  incu- 
bator for  24  hours  previous  to  inoculation. 


EXERCISE  87.  TUBERCLE  GROUP, 


213 


Bacterium  tuberculosis  (Kocn)  MIG. 
SYNONYMS.     Tubercle  bacillus;  Bacillus  tuberculosis  KOCH. 

EXPLANATORY.  First  described  by  Koch  in  1882.  Found  in 
diseased  tissues  of  man  and  animals  and  in  phthisical  sputum. 

REFERENCES.  Koch,  Berlin.  Klin.  Wochenschr.,  1882.  15,  221 ; 
Smith,  Jour.  Exp.  Med.,  1898,  3,  451 ;  A.  330 ;  C.  356 ;  PI.  2,  481 ; 
H.  225 ;  K.  &  W.  II,  78 ;  L.  &  K.  251 ;  L.  &  N.  410 ;  Mig.  2,  492 ;  M.  & 
R.  236 ;  M.  &  W.  148  ;  McF.  305  ;  P.  623. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet 

b.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT  Y  : 

a.  Character  of  movement 

b .  Flagella  stain , 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 4 


214  CULTURE  CHARACTERS 

Bact.  tuberculosis  does  not  grow  upon  the  ordinary  artificial 
media,  but  may  be  grown  upon  human  and  beef  blood  serum,  and 
after  it  has  been  isolated  for  some  time  it  may  be  grown  upon  bouil- 
lon, agar  and  potato  to  which  5%  of  glycerine  has  been  added. 
Media  which  are  suitable  for  the  isolation  of  this  organism  and  at 
the  same  time  are  easy  to  prepare  are  Smith's  dog  blood  serum 
(Jour.  Exp.  Med.,  1898,  3,  456),  and  Dorset's  Egg  Medium.  The 
last  is  the  simpler,  is  very  satisfactory,  and  is  prepared  as  follows : 
Perfectly  fresh  eggs  are  taken,  shell  broken  at  one  end  and  the  entire 
contents  poured  into  a  wide  mouthed  sterile  flask.  The  yolks  are 
broken  with  a  sterile  platinum  wire  and  25  cc.  of  water  added  to 
each  four  eggs,  and  then  the  flask  is  shaken  until  the  contents  are 
evenly  mixed.  The  mixture  is  then  strained  through  sterile  cloth 
which  removes  the  bubbles  and  makes  a  homogeneous  medium.  Run 
into  sterile  test-tubes,  about  10  cc.  in  each,  and  incline  in  a  serum 
inspissator  or  oven  and  heat  up  to  70°  C.  until  coagulated.  This 
usually  requires  four  to  five  hours  a  day  for  two  days.  This  is  all 
of  the  sterilization  usually  needed.  If  heated  higher  the  medium 
is  hardened  quicker  but  the  tubercle  germ  does  not  seem  to  grow  so 
rapidly.  Before  inoculating  the  tubes  they  should  be  sealed  and 
placed  in  the  incubator  for  several  days.  Cultures  from  tubercular 
lesions  are  made  by  tearing  the  tubercle  out  with  sterile  forceps, 
crushed  as  well  as  possible  with  the  forceps,  transferred  to  the  egg- 
slopes  with  a  sterile  platinum  loop ;  leave  bits  of  tissue  on  medium, 
avoid  breaking  surface  of  medium.  (Amer.  Med.,  1902,  and  Bull. 
52,  Part  I.,  Bureau  of  Animal  Industry,  1904.)  The  tubercle  bac- 
terium is  very  sensitive  to  temperature  variations  and  should  there- 
fore be  kept  at  a  temperature  varying  at  most  only  a  degree  or  two 
from  38°  C.  It  is  also  extremely  sensitive  towards  desiccation,  and, 
for  this  reason,  the  cotton  plug  should  be  well  paraffined,  or  replaced 
by  a  cork  through  which  a  small  cotton-plugged  glass  tube  passes, 
and  the  atmosphere  of  the  incubator  kept  saturated  with  moisture. 


BACTERIUM  TUBERCULOSIS  215 


216 


TUBERCLE  GROUP— CONTINUED. 


Bacterium  tuberculosis  var.  avium  (KRUSE)  MIG. 

SYNONYMS.  Bacillus  of  fowl  tuberculosis;  Bacillus  tuberculosis 
avium  KRUSE. 

EXPLANATORY.  This  organism  was  first  separated  from  B. 
tuberculosis  by  Maffucci,  and  is  probably  only  a  variety  of  the  latter. 
It  is  pathogenic  for  fowl,  but  ordinarily  not  for  other  animals. 

REFERENCES.  Maffucci,  Z.  fur  H.,  1892,  11,  445 ;  C.  356 ;  K.  & 
W.  II,  127  ;  L.  &  N.  418  ;  Mig.  2,  495. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

l>.  Agar 

c.  Gelatin 

d .  Other  media * 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 
6-  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 
6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d,  PleomorDhism 


BACTERIUM  TUBERCULOSIS  var.  AVIUM  217 

Culture  requirements  practically  the  same  as  for  Bact.  tuber- 
culosis. 


218  CULTURE  CHARACTERS 


EXERCISE  88.     COLON  GROUP. 


Bacillus  coli  (ESCHERICH)  MIG. 
SYNONYMS.     Bacterium  coli  commune  ESCH.  ;  Colon  bacillus. 

EXPLANATORY.  Escherich  was  the  first  to  describe  this  organism 
which  is  widely  known  as  a  common  inhabitant  of  the  colon  of  man, 
and  of  some  of  the  lower  animals. 

REFERENCES.  Escherich,  Darmbakt.  des  Sauglings,  Stuttgart, 
1886  ;  A.  432  ;  C.  205  ;  H.  282  ;  K.  &  W.  II,  334  ;  L.  &  N.  243  ;  Mig.  2, 
734  ;  M.  &  R.  325  ;  McF.  510. 


MORPHOLOGICAL  CHARACTERS  : 


!  SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 

5.  Loeffier's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT  Y  : 

a.  Character  of  movement.. 

6.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles..   . . 

d.  Pleomorphism 


CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -[-   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48hoursat °C.  6  days  at 


.'C. 


(a)  Surface  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at CC. 


Special  Media:    (Such  a«  litmus  milk  and  blood  serum.) 


BACILLUS  COLI 


221 


Gelatin  Stab:    Grown  24  hours  at °C. 


11 


48  hours  at »C.  6  days  at oC. 

Agar  Streak:    Grown  24  hours  at °C.  ^  Q 

til/          iv 

os  w 

?v   jl 

jyj 

48  hours  at °C.  6  days  at °C. 

Potato:    Grown  24  hours  at °C.  y 

o 

i 

1 

48  hours  at ...  1 °C.  6  days  at °C 

Bouillon:    Grown  24  hours  at °C.  ^  Q 

ts  is 

£  2 

1          I 

48hoursat °C.  6  days  at ...°C 


222  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: , 

optimum °C.;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: c 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent.,  48  hours percent 

72  hours per  cent hours per  cent 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatie. . .  


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


COLON  GROUP— CONTINUED. 


223 


Bacillus  enteritidis  GAERTNER 

EXPLANATORY.  Isolated  by  Gaertner  from  beef  in  meat  poison- 
ing case,  very  closely  related  to  preceding. 

REFERENCES.  Gaertner,  Correspond,  d.  allg.  Artze  Vereins, 
Thuringen,  1888 ;  C.  207 ;  Fl.  2,  375 ;  K.  &  W.  II,  639 ;  L.  &  N.  251 ; 
Mig.  2,  744 ;  M.  &  K.  331 ;  McF.  517. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  grentian- violet.. 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


224  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)         .  . 


or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at *C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  ENTERITIDIS 


225 


Gelatin  Stab:     Grown  24  hours  at °C. 


48  hours  at 


6  days  at 


Agar  Streak:     Grown  24  hours  at °C. 


!  I! 


48  hours  at °C.  6  days  at °C. 

Potato:    Grown  24  hours  at °C. 

/\ 

/      MX 

£ 
[ 

V 

i 

\ 

\ 

48  hours  at °C.  6  days  at °C. 

Bouillon:     Grown  24  hours  at. 


48  hours  at  ....................  °C. 


6  days  at  .......................  °C. 


226  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk . 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  89.  HOG  CHOLERA  GROUP. 


227 


Bacillus  Salmonii  (TREVISAN)  CHESTER. 

SYNONYMS.  Hog-cholera  bacillus;  B.  suipestifer  KRUSE;  Bact. 
cholera-suum  LEHM.  &  NEUM. 

EXPLANATORY.  First  described  by  Klein,  1884,  first  cultivated 
by  Salmon  and  Smith  in  1885.  Occurs  in  blood,  organs  and  intes- 
tinal contents  of  hogs  suffering  from  hog  cholera. 

REFERENCES.  Salmon  and  Smith,  Kept.  Bureau  Anim.  Ind., 
1885-91 ;  C.  210 ;  H.  281 ;  K.  &  W.  Ill,  622  ;  L.  &  N.  252 ;  Mig.  2,  759  ; 
McF.  538. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

i 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles , 

d.  Pleomorphism 


228  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  4.   . , or  —  . 

Gelatin  plate:    Grown  24  hours  at.... °C.  Sketches. 

(a)  Surface  Colonies.  :   (b)  Deep  Colonies. 

I 
48hoursat °C.  6  days  at 

Agar  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  ;   (b)  Deep  Colonies. 

48  hours  at °C.  6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  SALMONII 


229 


Gelatin  Stab:    Grown  24  hours  at. 


Agar  Streak:    Grown  24  hours  at °C. 


O 


48  hours  at  °C. 

6  days  at  .        .          ....           0C 

48  hours  at °C. 


6  days  at °C. 


Potato :    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at °C. 


230  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 

3.  RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— , 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.,  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: .  . 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


HOG  CHOLERA  GROUP—  CONTINUED. 
Bacillus  icteroides  SANARELLI. 


331 


EXPLANATORY.  First  described  in  1897  by  Sanarelli,  and  claimed 
by  him  to  be  the  cause  of  yellow  fever.  Very  closely  related  to 
preceding. 

REFERENCES.  Sanarelli,  Ann.  d.  PInst.  Past,  1897;  L.  &  N. 
256 ;  M.  &  R.  456 ;  McF.  525 ;  P.  609. 


MORPHOLOGICAL  CHARACTERS 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

6.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles , 

d .  Pleomorphism 


232  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  _|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  ;   (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  ICTEROIDES 


233 


Gelatin  Stab:    Grown  24hours  at °C. 


48  hours  at 


H'. 


6  days  at 0C. 


Agar  Streak:    Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 


48  hours  at 


'C. 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at, 


234  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: « 

optimum °C.;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  EELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc-:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

.  (2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent, 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION : . . .  ....... 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days , 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  90.     TYPHOID  GROUP. 


235 


Bacillus  typhosus  ZOPF. 

SYNONYMS.  Typhoid  bacillus;  Eberth's  bacillus ;  Bacillus  typhi 
ibdominalis  AUT. 

EXPLANATORY.  First  described  by  Eberth  in  1880,  first  culti- 
vated by  Gaffky,  1884.  It  is  found  in  the  feces  and  urine  of  typhoid 
patients. 

REFERENCES.  Eberth,  Virchow's  Archiv.  1880,  81,  58  and  1881, 
83,  486  ;  Gaffky,  Mitth.  aus  dem  Kaiserlichen  Gesundheitsamte,  1884, 
2,  372 ;  A.  408 ;  C.  213 ;  H.  263 ;  K.  &  W.  II,  204,  166 ;  L.  &  N.  232 ; 
Mig.  727 ;  M.  &  R.  319 ;  McF.  481 ;  P.  402. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


T.    FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Othermedia 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

I.    MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


236  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  -j- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies .  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


(a)  Surface  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

:   (b)  Deep  Colonies. 


Sketches. 


48  hours  at  

oC 

«c. 

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  TYPHOSUS 


237 


Gelatin  Stab:    Grown  24  hours  at °C. 


Agar  Streak;    Grown  24  hours  at 


v 


6  days  at                                       oC 

•C. 


48  hours  at 


6  days  at °C. 


Potato:    Grown  24  hours  at °C. 


48  hours  at . . 


6  days  at °C. 


Bouillon:     Grown  24  hours  at. 


V 


48  hours  at °C. 


6  days  at. , 


238  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure .minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  ligrht,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5-      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent, 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

b.  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION : 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


TYPHOID  GROUP— CONTINUED.  239 

Bacillus  dysenteriae  SHIGA. 
SYNONYM.     Bacillus  of  Japanese  dysentery,  SHIGA. 

EXPLANATORY,  First  described  by  Shiga,  who  found  it  causally 
related  to  a  dysentery  epidemic ;  Flexner  has  more  recently  found  it 
in  the  Philippines  and  elsewhere. 

REFERENCES.  Shiga,  C.  f .  B.,  1898,  23  ;  599  and  24 :  817,  870  and 
913 ;  Eldridge,  Public  Health  Repts.,  1900,  15 ;  p.  1,  Flexner,  Phil. 
Med.  Jour.  1900,  Sept.  1 ;  A.  440 ;  C.  228.  K.  &  W.  II,  317 ;  M.  &  R. 
350 ;  McF.  519. 

MORPHOLOGICAL  CHARACTERS:  i  SKETCHES. 

1.    FORM  AND  ARRANGEMENT: 

a.  Bouillon 


b.  Agar.. 


.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement. . 
6.  Flagella  stain 


SPORES: 


f.    SPECIAL  CHARACTERS: — 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism 


240  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -J- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a,)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »0. 


Agar  plate:    Grown  24  hours  at °C. 

t)  Surf  ace  Colonies.  (b)  Deep  Colonu 


Sketches. 


48  hours  at  

°C 

6  days  at  

Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  DYSENTERIAE 


241 


Gelatin  Stab:    Grown  24  hours  at.*. °C. 


!    - 


48  hours  at 


6  days  at oC. 


Agar  Streak:     Grown  24  hours  at °C. 


48  hours  at . . 


6  days  at °C. 


Potato:    Grown  24  hours  at 


48  hours  at  °C 

6  days  at                                       °C 

Bouillon:     Grown  24  hours  at °C. 


48  hours  »t -C. 


6  days  at °C, 


242  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: « „. 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION:. 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: , 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.,  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H :  CC«2:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION : . . . 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein, 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters ):., 


TYPHOID  GROUP— CONTINUED. 
Bacillus  pestis  (LEHM.  &  NEUM.)  CHESTER. 


S43 


SYNONYMS.  Bacterium  pestis  LEHM.  &  NEUM.  ;  Bacillus  pestis- 
bubonicae  KRUSE  ;  Bacillus  of  bubonic  plague. 

EXPLANATORY.  Described  at  about  the  same  time  independently 
by  Kitasato  and  Yersin  in  1894.  Found  in  the  buboes,  and  occa- 
sionally in  the  feces,  urine  and  blood  and,  in  the  pneumonic  form, 
in  the  sputum. 

REFERENCES.  Kitasato,  Lancet,  1894,  2,  428 ;  Yersin,  Ann.  Inst. 
Past.,  1894,  8,  662  ;  A.  310  ;  C.  215  ;  H.  291 ;  K.  &  W.  II,  475  ;  M.  &  R. 
435  ;  L.  &  N.  213  ;  Mig.  2,  749  ;  McF.  559  ;  P.  606. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

a.  Other  media 

2.  SIZE  : 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet 

b.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT  Y : 

a.  Character  of  movement 

&.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS  : , 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

<l.  Pleomorphism 


244  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller 's  scale)  -|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at ,  ...°C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at ° C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  PESTIS 


245 


Gelatin  Stab:    Grown 24hours  at °C. 


48  hours  at  ....................  °C. 


6  days  at 0C. 


Agar  Streak:     Grown  24  hours  at  ................  °C. 


48  hours  at °C.  6  days  at 

Potato:    Grown  24  hours  at °C. 

C3 

48  hours  at °C.  6  days  at °C. 

Bouillon:    Grown  24  hours  at °C. 

05 

2 
48  hours  at . . . . °C.  6  days  at...  ...«C. 


24G  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : . . . 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5-      GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

6.  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION:... ...... 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: , 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  91.     PSEUDOMONAS  GROUP. 


247 


Pseudomonas  aeruginosa  (SCHROETER)  MIG. 

SYNONYMS.  Bacillus  pyocyaneus  GESSARD;  Bacillus  of  blue- 
green  pus. 

EXPLANATORY.  First  described  in  1872  by  Schroeter.  Found 
in  green  pus,  and  widely  distributed  in  nature. 

REFERENCES.  Schroeter,  Cohn's  Beitraege  zur  Biologie,  1872,  1> 
126;  Barker,  Jour.  Am.  Med.  Asso.,  1897,  July  31;  Jordan,  Jour. 
Exp.  Med.,  1890,  627 ;  Lartigau,  IMd.,  1898,  595 ;  A.  304 ;  C.  321 ; 
H.  171 ;  K.  &  W.  Ill,  471 ;  L.  &  N.  281 ;  Mig.  884 ;  M.  &  R.  186  ;  M.  & 
W.  160 ;  McF.  269  ;  P.  535  ;  S.  454. 

MORPHOLOGICAL  CHARACTERS-.  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE  : 

3.  STAINING  POWE.RS  : 

a.  Aqueous  gentian- violet 

b.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement 

b.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism • 


248  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -|-  or  —  . 

Gelatin  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48hoursat °C.  6  days  at CC. 

Agar  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48  hours  at °C.  6  days  at 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


PSEUDOMONAS  AERUGINOSA 


249 


Gelatin  Stab:    Grown  24  hours  at °C. 


4S  hours  at °C. 


6  days  at «C. 


Agar  Streak:     Grown  24  hours  at 


Q 


48  hours  at 


6  days  at.. 


Potato:     Grown  24  hours  at , 


48  hours  at  . 


6  days  at.. 


Bouillon:     Grown  24  hours  at. 


48  hours  at °C. 


6  days  at. 


250  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C.;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minute*; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS... 
desiccation,  light,  disinfectants,  etc. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: , 


litmus  milk, 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteoly  tic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  92.  CHOLERA  GROUP. 


251 


Microspira  comma  (KOCH)  SCHROETER. 

SYNONYMS.  Spirillum  cholerae-asiaticae  ZOPF  ;  Vibrio  cholerae 
LEHM.  &  NEUM.  ;  Comma  bacillus ;  Cholera  bacillus. 

EXPLANATORY.  First  described  by  Koch  in  1884.  Found  in 
the  intestinal  contents  of  cholera  patients.  It  has  also  been  isolated 
several  times  from  water  supplies. 

REFERENCES.  Koch,  Berl.  Klin.  Wochenschr.,  1884,  no.  31  u.  32 ; 
A.  446  ;  C.  335  ;  Fl.  2,  527  ;  H.  333  ;  K.  &  W.  Ill,  1 ;  L.  &  N.  353 ;  Mig. 
2,  960  ;  M.  &  R.  407 ;  M.  &  W.  152  ;  McF.  442  ;  P.  568. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE:  

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet 

b.  Loeffler's  methylen-blue , 

c.  Gram's  stain , 

d.  Special  stains , 

4.  MOTILITY  : 

a.  Character  of  movement 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

&.  Involution  forms > 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


252  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller 's  scale)  _}_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


MICROSPIRA  COMMA 


253 


Gelatin  Stab :    Grown  24  hours  at °C. 


o 


48  hours  at °C.  6  days  at 

Agar  Streak:     Grown  24  hours  at °C.  Q  p 

•  _ 

"i    \i 

\ 

\ 

48  hours  at °C.  6  days  at °C 

Potato:     Grown  24  hours  at °C.  p  o 

^X  47  ^ 

c 

48  hours  at °C.  6  days  at °C. 

Bouillon:    Grown  24  hours  at °C.  rj  Q 


V 


48  hours  at °C. 


6  days  at °C. 


254  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  ligrht,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2-.  : : „ 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteoly tic 

digestion  of  gelatin digestion  of  casein. 

diastatic • 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


CHOLERA  GROUP— CONTINUED. 


255 


Microspira  Metschnikovi  (GAMALEIA)  MIG. 
SYNONYM.     Vibrio  Metschnikovi  GAMALEIA. 

EXPLANATORY.  First  described  in  1888  by  Gamaleia.  Fonnd 
in  intestinal  contents,  in  blood  and  in  organs  of  chickens  suffering 
from  a  disease  resembling  chicken  cholera. 

REFERENCES.  Gamaleia,  Ann.  Inst.  Past.,  1888,  2,  482 ;  A.  485 ; 
C.  334 ;  H.  345 ;  K.  &  W.  Ill,  68 ;  L.  &  N.  366 ;  Mig.  2,  979 ;  M.  &  K. 
427  ;  McF.  462 ;  P.  593. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: , 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c.  Deposits  or  vacuoles , 

d.  Pleomorphism 


256  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  _|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


• 

0£« 

«c 

A  gar  plate:    Grown  24  hours  at. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


MICROSPIRA  METSCHNIKOVI 


257 


Gelatin  Stab:    Grown  24  hours  at. 


SC. 


48  hours  at 


6  days  at 


Agar  Streak.:     Grown  24  hours  at 


48  hours  at °C. 


6  days  at. 


Potato:     Grown  24  hours  at 


Bouillon:     Grown  24  hours  at. 


48  hours  at  °C 

6  days  at                                         °C 

48  hours  at °C. 


6  days  at.. 


258  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C.   limits to °C. ; 

thermal  death-point °C.;  timeof  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: , 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  ?4  hours per  cent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CC»2:  : : 

1.  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION : , 


litmus  milk. 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . , 


CHOLERA  GROUP  — CONTINUED. 
Microspira  Schuylkilliensis  (ABBOTT)  CHESTER. 


259 


SYNONYM.     Vibrio  Schuylkilliensis  ABBOTT. 

EXPLANATORY.     Isolated  from  the  Schuylkill  river  water  by 
Abbott  in  1896.     Very  similar  to  preceding. 

REFERENCES.     Abbott,  Jour.  Exp.  Med.,  1896,  1,  p.  419 ;  A.  490 ; 
C.  334 ;  M.  &  R.  428 ;  McF.  465. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d .  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet. . 
ft.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS  : 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


260  CULTURE  CHARACTERS 

Keaction  of  media  (Fuller's  scale)  -- 


Gelatin  plate:    Grown  24  hours  at °C        j     Sketches 


(a)  Surface  Colonies. 


(b)  Deep  Colonies,  j 


48  hours  at °C. 


6  days  at "C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


4b  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


MlCROSPIRA   SCHUYLKILLIENSIS 


261 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at . . 


6  days  at 0C. 


Agar  Streak:     Grown  24  hours  at , 


O 


48  hours  at 


6  days  at °C. 


Potato:     Grown  24  hours  at °C. 


48  hours  at 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C.  6  days  at °C. 


262  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE  : 

optimum °C. ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes ; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FKEB  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COa:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk . 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein, 

diastatic ... 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :. 


EXERCISE  93.     STREPTOTHRIX  GROUP. 


263 


Streptothrix  bovis  (HARZ)  CHESTER. 

SYNONYMS.  Actinomyces  bovis  HARZ;  Streptothrix  actino- 
myces  Rossi-DoRiA;  Oospora  bovis  SAUV.  et  RADAIS;  ray  fungus; 
actinomyces. 

EXPLANATORY.  First  described  by  Bellinger.  It  occurs  in 
actinomycosis  or  lumpy- jaw  in  cattle,  hogs,  horses  and  man.  It 
probably  leads  a  saprophytic  life  on  plants,  etc. 

REFERENCES.  A.  361 ;  C.  361 ;  H.  349 ;  K.  &  W.  II,  861 ;  L.  &  N. 
440  ;  M.  &  R.  287  ;  McF.  371. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet.. 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY:.. 

a.  Character  of  movement.. 
6.  Flagella  stain 

5.  SPORES  : 

<5.    SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism , 


264  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller 's  scale)  4.   or  — 


Gelatin  plate:    Grown  24  hours  at °C.       j     Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


48  hours  at °C. 


6  days  at *C 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


STREPTOTHRIX  Bovis 


265 


Gelatin  Stab:    Grown  24  hours  at °C. 


•iS  hours  at 


days  at . 


Agar  Streak:     Grown  24  hours  at °C. 


6  days  at.. 


Potato:     Grown  24  hours  at 


C. 


48  hou  rs  at 


°C.  6  days  at  .......................  °C. 

I 


Bouillon:    Grown  24  hours  at      °C                r^ 

ta 
h 
J3 
O 

«S 
O 

~^_J 

^J 

48  hours  at  °C.                                 6  days  at... 

0 

n. 

266  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C.  • 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:.... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION:. 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  :  : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :. 


STREPTOTHRIX  GROUP— CONTINUED. 


267 


Streptothrix  Madurae  VINCENT. 

EXPLANATORY.     First  described  by  Vincent.     Associated  with  a 
warty,  ulcerative  affection  of  the  feet,  but  rarely  of  the  hands. 

REFERENCES.     Vincent,  Ann.  Past.  Inst.,  1894;  A.  365;  C.  368; 
H.  356 ;  K.  &  W.  II,  839,  III,  454 ;  L.  &  N.  452 ;  M.  &  R.  297 ;  McF. 

378. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Othermedia 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL,  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

(1.  Pleomorphism 


268  CULTURE  CHARACTERS 

Keaction  of  media  (Fuller's  scale)  -|-  or  —  . 

Gelatin  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48hoursat °C.  6days  at «C. 

Agar  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

4 
48  hours  at °C.  6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


STREPTOTHRIX  MADURAE 


269 


Gelatin  Stab*    Grown  24  hours  at            •  •           °C                r^ 

O 

0 

ts 

"S 

o 

0 

V           / 

• 

V    j 

48  hours  at 


6  days  at 


Agar  Streak:    Grown  24  hours  at , 


I 


48  hours  at 


6  days  at. 


Potato:     Grown  24  hours  at 


48  hours  at 


6  days  at.. 


Bouillon:    Grown  24  hours  at. 


48  hours  at "C. 


6  days  at. 


270  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °  C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent.,  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

b,  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: .  . 


proteolytic 

• 
digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


MEDICAL  BACTERIOLOGY 


271 


CHAPTER  VII 
PATHOGENIC  ANAEROBES 


Anaerobic  bacteria  may  be  furnished  conditions  which  permit  of 
their  development,  in  a  variety  of  ways,  and  a  very  considerable 
number  of  pieces  of  apparatus  have  been  devised  to  secure  this  end. 
In  a  general  way  all  of  the  methods  may  be  grouped  under  the 
following  heads : 

1.  Displacement  of  air. 

2.  Absorption  of  oxygen. 

3.  Exhaustion  of  air. 

4.  Exclusion  of  air. 

5.  Miscellaneous  methods,  in  the  presence  of  reducing  substances 
as  litmus,  or  a  strongly  aerobic  germ,  etc. 

The  first  two  methods  are  the  most  reliable.  In  the  displace- 
ment method,  hydrogen,  carbon  dioxide  or  illuminating  gas  may 
be  used;  hydrogen  is  best.  This  gas  is  readily  prepared  by  the 
action  of  sulphuric  acid  (1:8) 
on  zinc.  Either  a  Kipp  gen- 
erator may  be  used  or  one  of 
a  simpler  construction  (Fig. 
32).  The  gas  should  be 
washed,  1st,  in  lead  nitrate 
to  absorb  the  sulphuretted 
hydrogen,  2d,  in  silver  sul- 
phate to  absorb  any  arseniur- 
etted  or  phosphuretted  hydro- 
gen, and  3d,  in  potassium  hy- 
drate to  remove  sulphur  and 
carbon  dioxide. 

The  cultures  should  be  made  in  dextrose  media  (which  should 
preferably  be  freshly  prepared  and  always  boiled  immediately  be- 
fore being  inoculated),  either  as  test-tube  or  plate  cultures.  Novy's 
anaerobic  jars  are  perhaps  the  most  satisfactory  receptacles  for  the 
cultures.  (For  description  of  same,  see  N.  306.) 


FIG.  32.    Hydrogen  Generator. 


272 


MEDICAL  BACTERIOLOGY 


In  the  second  method  (Buchner's  method)  an  alkaline  solution 
of  pyrogallic  acid  is  used  to  absorb  the  oxygen.  The  cultures  may 
be  placed  in  Novy  jars  or  similar  receptacles;  for  tube  cultures  a 
large  wide  mouthed  bottle  fitted  with  a  rubber  cork  does  very  well. 
The  dry  pyrogallic  acid  is  placed  in  the  bottom  of  the  receptacles, 
about  1  gram  to  every  100  cc.  of  air  space,  the  tubes  are  put  in  place, 
then  about  10  cc.  of  a  normal  sodium  hydroxide  solution  are  added 
to  each  gram  of  pyrogallic  acid,  and  the  apparatus  immediately  and 
hermetically  sealed.  A  very  convenient  method  has  recently  been 
outlined  by  Wright  for  test-tube  cultures.  It  is  as  follows :  After 
the  culture  medium  has  been  inoculated  in 
the  usual  manner,  thrust  the  cotton  plug 
into  the  test-tube  so  that  the  upper  end  of 
the  cotton  is  about  2  cm.  below  the  mouth 
of  the  test-tube  (it  is  usually  desirable  to 
cut  off  a  part  of  the  protruding  portion 
before  doing  this).  Fill  the  tube  with  py- 
rogallic acid.  Add  with  a  pipette  enough 
of  a  4%  solution  of  sodium  hydrate  to  dis- 
solve the  acid.  Close  the  tube  immediately, 
making  it  air  tight  by  inserting  a  rubber 
stopper  in  its  mouth.  Then  invert,  in  the 
case  of  solid  media,  and  set  aside  for  devel- 
opment. Fig.  33.  Rickards  has  recently 
published  a  modification,  which  consists  in 
inverting  the  inoculated  tubes,  without  the 
plugs,  into  a  glass  in  which  is  a  layer  of  dry 
pyrogallol  and  then  adding  the  hydroxide.  Plate  cultures  are  made 
by  using  Erlenmeyer  flasks  instead  of  Petri  dishes. 

REFERENCES.  A.  206 ;  L.  &  K.  98 ;  M.  &  R.  68 ;  M.  &  W.  117 ; 
McF.  216  -P.  233 ;  S.  78 ;  Wright,  Jour.  Boston  Soc.  of  Med.  Sci., 
1900,  5,  114 ;  Rickards,  C.  f .  B.,  1st  Abt,  Originale,  36 ;  557. 


FIG.  33.  Wright's  method 
for  cultivating  anaerobes, 
a,  cotton  plug;  b,  alkaline 
pyrogallic  acid  solution;  c, 
rubber  cork. 


EXERCISE  94.     EMPHYSEMA  GROUP.  273 

Bacterium  Welchii  MIG. 

SYNONYMS.  Bacillus  aerogenes  capsulaius  WELCH;  B.  der  Gasphlegmon 
FRAENKEL;  B.  emphyseraatasus  KRUSE. 

EXPLANATORY.  First  described  by  Welch  in  1892.  Occurs  at  autopsies  in. 
which  gas  bubbles  are  present  in  the  larger  vessels,  accompanied  by  the  for- 
mation of  numerous  small  cavities  in  the  liver  containing  gas.  It  has  been 
found  also  in  emphysematous  phlegmons,  in  puerperal  sepsis,  in  peritonitis 
and  in  other  conditions  (M.  &  W.).  Widely  distributed  in  nature.  (Welch.) 

REFERENCES.  Welch  and  Nuttall,  Bull.  Johns  Hopkins  Hospital,  1892,  3, 
81;  Welch  and  Flexner,  Jour.  Exp.  Med.,  1896,  1,  5;  C.  183;  H.  329;  L.  &  N. 
344;  Mig.  392;  M.  &  R.  402;  McF.  591;  P.  545. 


MORPHOLOGICAL  CHARACTERS:  j  SKETCHES. 

1.    FORM  AND  ARRANGEMENT: 

«.  Bouillon : 


b.  Agrar 


c.  Gelatin 


L  Other  media. 


SIZE: 

STAINING  POWERS  : 

a.  Aqueous  gentian-violet.. 

b.  Loeffler's  methylen-blue. 


\ 
c.  Gram's  stain 


d.  Special  stains 

4.    MOTILIT  Y  : 

a.  Character  of  movement. 

b.  Flagella  stain 


5.    SPORES:... 


SPECIAL  CHARACTERS:.... 

a.  Capsules 

b.  Involution  forms 

o.  Deposits  or  vacuoles. 
d.  Pleomorphism 


274  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller  >s  scale)  -|_   or  — 


Gelatin  plate:    Grown  24  hours  at .°C. 


(a)  Surf  ace  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C.  6  days  at »C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACTERIUM  WELCHII 


275 


Gelatin  Stab:     Grown  24  hours  at °C. 


15 


48  hours  at                                   °C 

6  days  at                                       oC 

Agar  StreaK:     Grown  24  hours  at °C.  rj 


48  hours  at 


6  days  at °C. 


Potato:     Grown  24  hours  at °C. 


2  i 


48  hours  at 


6  days  at., 


Bouillon:     Grown  24  hours  at. 


48  hours  at «C.  6  days  at.. 


'C. 


276  PHYSIOLOGICAL  CHARACTERS 


1    RELATION  TO  TEMPERATURE: 

optimum °C.  •  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.    RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  ligrht,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: c. 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: . 


litmus  milk, 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic « 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :. 


EXERCISE  95.  OEDEMA  GROUP. 


277 


Bacillus  Feseri  (TREVISAIN)  KITT. 

SYNONYMS.  Bacillus  of  symptomatic  anthrax;  Black-leg  bacil- 
lus; Bacillus  carbonis  MIG.  ;  Bacillus  anthracis-symptomatici  KRUSE. 

EXPLANATORY.  First  described  by  Arloing,  Cornevin  and 
Thomas  in  1880.  It  occurs  in  the  subcutaneous  tissue,  muscles  and 
serous  exudate  of  animals  suffering  from  symptomatic  anthrax. 

KEFERENCES.  Arloing,  Cornevin  and  Thomas,  Le  Charbon 
symptomatique  du  boeuf ,  2nd  edit.  Paris,  1887  ;  A.  527 ;  C.  296 ;  Fl. 
2,  245  ;  H.  328  ;  K.  &  W.  II,  601 ;  L.  &  N.  339  ;  Mig.  593  ;  M.  &  R.  401 ; 
McF.  583 ;  P.  563. 

MORPHOLOGICAL  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE  : 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet 

ft.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT Y  : 

a.  Character  of  movement , , 

b.  Flagella  stain 

6.    SPORES  : 

6.    SPECIAL  CHARACTERS  : 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


278  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  _|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies,  (b)  Deep  Colonies. 


Sketches. 


48  hours  at. 


°C.  6  days  at. 


•C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at 'C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  FESERI 


279 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at 


Agar  Streak:    Grown  24  hours  at °C.  H  I 


I     \. 

V 


u  I 


48  hours  at 


6  days  at °C. 


Potato :    Grown  24  hours  at °C. 


6  days  at °C. 


Bouillon:     Grown  24  hours  at. 


48  hours  at . . 


O 


6  days  at °C. 


280  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum ° C. ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure .minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: ; 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours percent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

6.  lactose c.  saccharose 

6.      ACID  OR  ALKALI    PRODUCTION : .  .  .  


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days , 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :, 


OEDEMA  GrROOP  —  CONTINUED. 


281 


Bacillus  oedematis  ZOPF. 

SYNONYMS.  Bacillus  of  malignant  oedema;  Bacillus  oedematis- 
maligni  ZOPF. 

EXPLANATORY.  First  described  by  Pasteur  in  1877.  Widely 
distributed  in  soil  and  putrefying  material.  Few  cases  on  record 
of  infection  in  man. 

REFERENCES.  Zopf,  Spaltpilze,  1885,  88;  A.  522;  C.  292;  Fl.  2, 
234 ;  H.  326 ;  K.  &  W.  II,  619 ;  L.  &  N.  341 ;  Mig.  604 ;  M.  &  R.  393 ; 
Mc.F.  587  ;  P.  543  ;  S.  488. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

«.  Bouillon , 

&.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 

b.  Flagella  stain 

5.  SPORES  : 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles 

d.  Pleomorphism 


282  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  _|_   or  —  . 

Gelatin  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 

48 hours  at °C.  6 days  at , ...,o....*C. 

Agar  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


48  hours  at. *C. 


6  days  at. «C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  OEDEMATIS 


283 


Gelatin  Stab:    Grown  24  hours  at °C. 


Agar  Streak:     Grown  24  hours  at °C. 


Potato:    Grown  24  hours  at °C. 


Bouillon:     Grown  24  hours  at. 


48  hours  at °C. 


6  day* 


is 
e 


6  days  at         .                              oC 

48  hours  at                                   °C 

6  days  at                  °C 

'C. 


284  PHYSIOLOGICAL  CHARACTERS 


1«    RELATION  TO  TEMPERATURE: , 

optimum °C  ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.    RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.: — . 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours .per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CO2:  : : 

&.  lactose c.  saccharose 

6.     ACID  OR  ALKALI    PRODUCTION:...  .    


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION:...  .  . 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


OEDEMA  GROUP  — CONTINUED. 
Bacillus  footulinus  v.  ERMENGEM. 


285 


EXPLANATORY.  Isolated  by  v.  Ermengem  from  ham  which  had 
caused  meat  poisoning.  Believed  to  be  the  cause  of  meat  poisoning 
characterized  by  nervous  symptoms  of  central  origin,  botulism. 

REFERENCES,  v.  Ermengem,  Z.  f .  H.,  1898,  26,  1 ;  C.  297 ;  K.  & 
W.  II,  671 ;  L.  &  N.  337 ;  Mig.  616 ;  M.  &  R.  398. 

MORPHOLOGICAL,  CHARACTERS:  SKETCHES. 

1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon.. 

6.  Agar 

c.  Gelatin 

d.  Other  media. 

2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian-violet 

6.  Loeffler's  methylen-blue 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: 

a.  Character  of  movement. * 

6.  Flagella  stain 

5.  SPORES: 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms ,..,, 

c.  Deposits  or  vacuoles... i •••••••••••••••« ....ft 

d.  Pleomorwhism..... , ..,...»... ^.......,.................  ,, 


286  CULTURE  CHARACTERS 

Reaction  of  media  (Fuller's  scale)  _j_   or 


Gelatin  plate:    Grown  24  hours  at °C.  Sketches. 

(a)  Surface  Colonies,  (b)  Deep  Colonies. 


48  hours  at °C. 


6  days  at. 


(a)  Surf  ace  Colonies. 


Agar  plate:    Grown  24  hours  at °C. 

:   (b)  Deep  Colonies. 


•C. 


Sketches. 


48  hours  at °C. 


6days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


BACILLUS  BOTULINUS 


287 


Gelatin  Stab:    Grown  24  hours  at °C. 


Agar  Streak:     Grown  24  hours  at 


48  hours  at  °C 

6  days  at                                       0C 

o 


48  hours  at 


6  days  at.. 


Potato:     Grown  24  hours  at 


ts  y 


o 


48  hours  at . 


6  days  at °C. 


Bouillon:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at. , 


288  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE  : 

optimum °C. ;  limits to °C.; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc  : — . 


4.    PIGMENT  PRODUCTION; 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm. .  • closed  arm 

rate  of  de^7elopment:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CC-2:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: , 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: . 


proteolytic 

digestion  of  gelatin digestion  of  casein , 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


EXERCISE  96.  TETANUS  GROUP. 


289 


Bacillus  tetani  NICOLAIER. 

EXPLANATORY.  Discovered  by  Nicolaier,  1884.  First  cultivated 
by  Kitasato,  1889.  Occurs  in  man  and  in  animals  suffering  from 
the  disease,  and  is  widely  distributed  in  nature,  especially  in  soil. 

KEFERENCES.  Nicolaier,  Deutsche  Med.  Wochenschrift,  1884; 
Kitasato,  Deutsche  Med.  Wochenschrift,  1889  ;  A.  513  ;  C.  302  ;  Fl.  2, 
260 ;  H.  320 ;  K.  &  W.  II,  566 ;  L.  &  N.  332 ;  Mig.  592 ;  M.  &  R.  376 ; 
McF.  389 ;  P.  385. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.  FORM  AND  ARRANGEMENT: 

a.  Bouillon 

b.  Agar 

c.  Gelatin 

d.  Other  media 

2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian- violet. . 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains — 

4 .  MOTILJTY  : 

a.  Character  of  movement.. 
6.  Flagella  stain. . 

5.  SPORES  :...-  = , 

6.  SPECIAL  CHARACTERS: 

a.  Capsules 

&.  Involution  forms. ........ 

c.  Deposits  or  vacuoles , 

d.  Pleomorphism 


290  CULTURE  CHARACTERS 

Keaction  of  media  (Fuller's  scale)  _|_   or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  i  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at "C. 


Agar  plate:    Grown  24  hours  at. 


(a)  Surface  Colonies. 


:   (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.; 


BACILLUS  TETANI 


291 


Gelatin  Stab:    Grown  24  hours  at °C. 


Agar  Streak:     Grown  24  hours  at , 


48  hours  at                 'C 

6  days  at                                        0C 

48  hours  at . 


6  days  at °C. 


Potato:     Grown  24  hours  at , 


Bouillon:     Grown  24  hours  at. 


48  hours  at  °C 

6  days  at                                         °C 

48  hours  at °C. 


6  days  at., 


292  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc-:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA : 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent.,  48  hours percent. 

72  hours per  cent hours percent. 

reaction  in  open  arm 

gas  formula,  H:  COa:  : : 

b.  lactose c.  saccharose 

6.  ACID  OR  ALKALI    PRODUCTION:... 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites  to  ammonia. . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGEXESIS  (or  other  special  characters) 


294  MEDICAL  BACTERIOLOGY 


CHAPTER  VIII 

ANIMAL  INOCULATION  AND   STAINING  OF 
BACTERIA  IN  TISSUE 


EXERCISE  97.     ANIMAL  INOCULATION. 

METHODS  OF  INOCULATION.  Animal  inoculation  is  practiced  to 
determine  the  pathogenic  properties  of  an  organism  and  also  the 
character  of  the  tissue  changes  produced.  The  animals  commonly 
used  are  white  mice  and  white  rats,  rabbits,  guinea  pigs  and  pigeons. 
Inoculations  are  usually  made  subcutaneously,  intraperitoneally 
or  intravenously,  and  in  special  cases  into  the  pleural  cavity,  brain, 
eye,  etc.,  etc.  Mice  require  a  holder,  the  inoculation  being  usually 
made  at  the  root  of  the  tail.  Other  animals  can  usually  be  held  by 
an  assistant. 

Subcutaneous.  The  place  selected  is  usually  the  abdominal  wall. 
Pigeons  are  inoculated  in  the  pectoral  muscles ;  the  hair  or  feathers 
should  be  removed  and  the  skin  washed  with  a  disinfectant,  e.  g.,  5% 
carbolic  acid. 

a.  For  liquids  a  sterilized  hypodermic  syringe  is  used.  A  fold 
of  the  skin  is  raised,  the  needle  of  the  syringe  inserted  and  the 
requisite  amount  of  material  injected. 

&.  For  solid  material  a  pocket  is  made  which  is  stitched,  or  sealed 
with  contractile  collodion,  after  the  material  is  introduced. 

Intraperitoneal.  Either  liquids  or  solid  material  may  be  intro- 
duced. 

a.  For  liquids.  The  seat  of  inoculation  is  prepared  as  above,  the 
syringe  needle  is  then  plunged  directly  into  the  peritoneal  cavity. 

6.  For  solid  material.  The  animal  is  anesthetized ;  the  hair  is 
clipped  or  shaved  from  a  portion  of  the  median  line,  about  half  way 
between  the  pubis  and  the  sternum ;  a  slit  is  made  in  the  skin  with 
sterile  instruments ;  the  smallest  possible  opening  is  made  along  the 
linea  alba  into  the  peritoneal  cavity  and  the  material  introduced; 
the  wound  closed  and  the  body  wall  and  the  skin  stitched  separately. 
It  is  hardly  necessary  to  add  that  the  whole  operation  is  carried  out 
under  the  most  strict  aseptic  precautions.  Collodion  sacs  are  intro- 
duced in  this  way. 


296 


MEDICAL  BACTERIOLOGY 


Collodion  Sacs.  The  use  of  the  collodion  sac  has  recently  become 
very  common  and  deserves  description  as  one  of  the  necessary  labor- 
atory procedures.  The  difficulty  in  making  these  sacs  has  been 
largely  overcome  by  recent  methods.  One  of  these  is  the  following : 
Small-sized  test-tubes  are  selected.  Thick  collodion  is  then  poured 
into  the  tube  to  a  depth  of  two  inches.  The  collodion  is  then 
poured  out  along  one  side  of  the  tube  into  another  tube  and  from  this 
one  to  another  and  so  on  until  the  required  number  is  obtained.  The 
desired  length  of  the  sac  can  be  secured  in  all  of  the  tubes  by  tipping 
and  rolling  them,  thus  bringing  the  collodion  into  contact  with  the 
glass  to  the  proper  height.  As  the  tubes  are  coated  they  are  placed, 
mouth  down,  in  a  wire  basket  or  test-tube  rack  as  indicated  in  Fig. 
34,  1 ) .  In  this  way  the  extra  collodion  drains  off  and  free  access  of 
air  dries  and  hardens  the  collodion,  leaving  a  thin  coat  covering  the 
inner  surface  of  the  tube.  The  thickness  of  the  coat  depends  on  the 
consistency  of  the  collodion.  A  ten  per  cent,  collodion,  in  equal 
parts  of  alcohol  and  ether,  makes  a  sufficiently  thick  coat  for  ordi- 
nary purposes.  The  drying  may  be  stopped  at  any  point  by  filling 


i 


Fig.  34.  Preparation  of  Collodion  Sac:  1,  Tube  inverted  to  allow  the  extra  collodion 
to  drain  off  and  the  film  to  air-dry.  2,  Sac  ready  for  sterilization;  (A)  Surgeon's  knot; 
(B)  Ends  of  cord;  (C)  Tongue  of  collodion.  3,  Sac  ready  to  be  inoculated  into  animal. 

the  tube  with  water  and  after  standing  a  few  minutes  the  collodion 
shrinks  and  the  sac  may  be  easily  removed.  The  sacs  are  then  filled 
from  one-fourth  to  three-fourths  full  with  bouillon.  They  are  then 
immersed  in  a  test-tube  of  the  medium,.  The  sacs  are  held  in  posi- 
tion in  the  test-tube  by  means  of  the  tongue  formed  by  the  collodion 
flowing  out  of  the  tube.  This  tongue  is  folded  over  the  lip  of  the 
tube.  (Fig.  34,  2,  C.)  Before,  however,  the  sac  is  put  into  the  test- 
tube  a  piece  of  cotton  or  silk  cord  is  placed  around  the  sac  near  the 


MEDICAL  BACTERIOLOGY 


top  and  held  in  position  by  means  of  a  surgeon 's  knot,  loosely  drawn. 
The  cord  should  be  quite  stout  so  that  the  sac  can  later  be  tightly 
closed.  The  ends  of  the  cord  are  brought  outside  of  the  tube  as 
shown  at  B,  Fig.  34,  2.  Sterilization  may  be  accomplished  either  in 
the  autoclave  or  by  means  of  the  intermittent  method  of  sterilization. 

The  medium  is  inoculated  by  means  of  the  platinum  needles  in 
exactly  the  same  way  in  which  tube  cultures  are  ordinarily  inocu- 
lated. The  sac  thus  inoculated  should  be  incubated  for  twenty- 
four  hours  and  if  the  medium  outside  of  the  sac  remains  clear  the  sac 
may  be  used.  Otherwise  it  would  be  discarded. 

The  tube  is  placed  in  a  tumbler  or  test-tube  rack.  The'  sac  is 
then  pulled  out  of  the  tube  until  the  cords  can  be  drawn  tight  so  as 
to  close  the  sac  and  securely  tied.  With  sterile  scissors  the  end  of 
the  sac  is  cut  off  a  few  millimeters  above  the  constriction.  If  there 
is  any  moisture  on  the  inside  of  the  sac  above  the  neck  this  must  be 
removed  with  sterile  filter  paper  and  then  a  few  drops  of  a  thin 
solution  of  collodion  is  placed  in  the  neck  so  as  to  hermetically  seal 
the  sac.  The  long  and  contaminated  ends  of  the  cord  are  now  cut 
off,  the  sac  dropped  back  into  the  test-tube,  and  the  cotton  stopper 
replaced.  (Fig.  34,  3.)  The  sac  is  now  ready  to  be  placed  in  the 
body  cavity  of  an  animal.  (Frost.) 

The  method  of  inoculation  is  especially 'useful  in  increasing  the 
virulence  of  attenuated  forms,  and  in  producing  immunity  in  ani- 
mals to  induce  the  agglutinating  and  lysogenic  properties  in  the 
blood. 

REFERENCES.  Harris,  C.  f .  B.  I.,  1902,  32 :  74 ;  Frost,  Proc.  Am. 
Pub.  H.  A.,  1903,  28,  p.  536. 

Intravenous  A  rabbit  is  generally  chosen  for  this  purpose  and 
the  inoculation  made  into  an  ear  vein.  Of  the  three  branches  of 
the  vena  auricularis  posterior,  the  ramus  lateralis  posterior  is  the 
smallest,  but,  due  to  the  fact  that  it  is  the  most  firmly  imbedded  in 
connective  tissue,  it  is  much  more  easily  entered  than  the  others. 
The  artery  forceps  (Fig. 
35,  a)  are  used  to  gorge  the 
vessel  and  are,  of  course, 
removed  before  the  mate- 
rial is  injected.  Avoid  the 
introduction  of  air,  which 
causes  immediate  death, 
and  keep  the  animals  un- 
der close  observation  for 

FIG.  35.    Dorsal  view  ot  right  ear  of  rabbit. 
One  hour.  a,  artery  forceps;  b,  syringe. 


300 


MEDICAL  BACTERIOLOGY 


Inoculation  into  Lymphatic  system.  Fluid  cultures,  or  suspen- 
sions of  bacteria,  can  be  injected  into  the  lymphatics  by  way  of  the 
testicles,  by  plunging  the  point  of  the  needle  into  the  substance  of 
the  testicle  and  injecting  the  desired  amount  of  fluid. 

Inoculation  into  the  Pleural  Cavity.  Where  necessary  the  needle 
is  introduced  into  the  pleural  cavity  between  the  ribs.  It  is  very 
difficult  to  perform  this  experiment  without  injuring  the  lung. 

Inoculation  into  the  Anterior  Chamber  of  the  eye.  Rarely  prac- 
ticed. The  eye  is  treated  with  a  few  drops  of  cocaine  (2%  solution) 
and  then  the  needle  is  inserted  through  the  cornea  just  in  front  of 
its  junction  with  the  sclerotic,  the  needle  passing  into  the  anterior 
chamber  in  a  plane  parallel  to  the  plane  of  the  iris. 

STERILIZATION  OF  INSTRUMENTS.  These  are  best  sterilized  by 
boiling  in  a  solution  of  soda  or  borax  for  15  minutes.  This  is  ac- 
complished in  an  especially  designed  apparatus  or  in  an  ordinary 
enamel  stew  pan.  In  case  of  emergencies  the  instruments  may  be 
dipped  in  benzine  or  alcohol  and  burned.  This  is  less  injurious  to 
the  instruments  than  heating  in  the  direct  flame. 
Use  blank,  p.  308  for  preservation  of  data. 

OBSERVATION  OF  INOCULATED  ANIMALS.  After  inoculation  the 
animals  should  be  placed  in  separate  cages,  or,  if  placed  together, 

they  must  be  described  or 
marked  so  as  to  be  easily  identi- 
fied. Fig.  36  shows  a  simple 
cage  made  of  galvanized  iron 
with  soldered  seams.  After  use 
it  is  sterilized  by  boiling  water 
in  it.  The  wire  door  is  covered 
with  a  cloth  to  prevent  the  too 
rapid  escape  of  steam. 

The  inoculated  animals  must 
be  kept  under  constant  observa- 


PIG.  36.    Wesbrook's  sterilizable,  gal- 
vanized-iron  animal  cage. 


tion  and  the  following  conditions  noted : 
a.  Temperature. 
~b.  Loss  of  weight. 

c.  Peculiar  position  in  cage. 

d.  Loss  of  appetite. 

e.  Condition  of  the  coat  or  hair. 

/.  Condition  of  the  secretions  of  the  air  passages,  conjunctiva 
and  kidneys ;  diarrhea  or  hemorrhage  from  the  bowels. 
g.  The  condition  of  the  seat  of  inoculation. 


302  MEDICAL  BACTERIOLOGY 

The  animals  should  be  fed  regularly,  weighed  at  the  same  hour 
each  day  and  the  temperature  taken  at  the  rectum. 

POST  MORTEM  EXAMINATION. 

Perform  -the  autopsy  as  soon  as  possible  after  death.     When 

delay  cannot  be  avoided,  place  the  animal  in  the  ice-chest  until  such 

time  as  is  convenient. 

A. 

a.  Inspect  externally  and  note  presence  and  .character  of  any 
lesion. 

Z>.  Sterilize  a  suitable  post-mortem  board  with  corrosive  sub- 
limate solution,  1  to  1000,  place  the  animal  belly  upwards  and 
tack  the  four  legs  fast  to  the  board. 

c.  Wash  the  surface  of  the  thorax  and  abdomen  with  corro- 
sive sublimate  solution,  make  an  incision  through  the  skin  at 
the  pubis,  introducing  one  blade  of  the  scissors,  and  extend  the 
incision  as  far  as  the  chin. 

d.  Carefully  dissect  the  skin  away  from  the  abdomen,  thorax, 
axillary,  inguinal,  and  cervical  regions,  and  fore  and  hind  legs, 
and  pin  it  to  the  board  as  far  as  possible  from  the  thorax  and 
abdomen.     It  is  from  the  skin  that  the  chances  of  contamination 
are  greatest. 

B.  All  incisions  from  now  on  are  made  with  sterilized  instruments. 

a.  Take  an  ordinary  potato-knife,  heat  it  quite  hot,  and  place 
it  on  the  abdomen  in  the  region  of  the  linea  alba  until  the  fascia 
begins  to  burn ;  the  knife  is  then  held  transversely  to  this  line 
over  the  center  of  the  abdomen,  making  two  sterilized  tracks 
through  which  the  abdomen  may  be  opened  by  crucial  incisions  • 
two  burned  lines  are  also  made  along  the  sides  of  the  thorax. 

~b.  Make  a  central  longitudinal  incision  from  the  sternum  to 
the  genitalia  with  sterile  scissors,  the  abdominal  wall  being  held 
up  with  sterilized  forceps,  or  a  hook,  to  prevent  the  viscera  being 
injured.  A  transverse  incision  is  made  in  a  similar  manner. 
Cut  through  the  ribs  with  strong  sterilized  scissors  along  the 
sterilized  tracks  on  the  sides  of  the  thorax,  when  the  whole  ante- 
rior wall  of  the  thorax  is  easily  lifted  and  entirely  removed  by 
severing  the  diaphragm  connections. 

.  c.  When  the  thoracic  and  abdominal  cavities  are  fully  exposed, 
a  careful  examination  of  the  organs  and  surroundings  is  made 
without  disturbing  them. 

Plates  (Petri-dishes)  or  roll  cultures  are  prepared  from  the 
blood,  liver,  spleen,  kidneys,  and  from  any  exudates  present. 


304  MEDICAL  BACTERIOLOGY 

The  method  is  as  follows : 

( 1 )  Heat  a  scalpel  and  scorch  a  small  surface  of  the  organ  from 
which  the  cultures  are  to  be  made. 

(2)  Heat  the  scalpel  again  and  penetrate  the  capsule  of  the 
organ  with  the  point,  and  through  the  opening  insert  a  stout  ster- 
ilized platinum  loop,  push  it  into  the  tissues,  twist  around,  and 
obtain  enough  material  from  the  center  of  the  organ  to  make  the 
culture.     Cultures  from  blood  are  usually  made  from  one  of  the 
heart  cavities,  the  surface  being  seared  with  a  hot  knife  before 
opening. 

As  soon  as  the  culture  material  is  obtained,  cover-glass  speci- 
mens are  prepared  from  each  organ  and  from  existing  exudates. 

Small  pieces  of  each  organ  are  also  preserved  for  future  exam- 
ination. 

When  the  autopsy  is  finished  the  remainder  of  the  animal  should 
be  burned1  and  the  instruments  should  be  sterilized  (see  p.  300). 
Wash  the  post-mortem  board  with  sublimate  solution.  The  cover- 
glasses  and  other  material  likely  to  contain  infectious  matter  must 
also  be  sterilized  when  of  no  further  use. 

Cultures  are  to  be  incubated  at  38°  C.,  growth  examined  micro- 
scopically, and  by  means  of  sub-cultures. 

Use  blank  on  p.  308  for  preservation  of  data.  Fig.  37  shows  the 
method  of  making  a  post-mortem  and  the  location  of  the  most  im- 
portant lymphatic  glands. 

REFERENCES.  The  above  is  taken  largely  from  Bowhill,  74 ;  see 
also  A.  230 ;  N.  260 ;  and  other  texts. 

COMMON  LABORATORY  EXPERIMENTS. 

The  following  inoculations  are  those  most  frequently  made : 

Streptococcus  erysipelatos.     Mice  or  rabbits,  intravenous. 

M.  pyogenes  var.  aureus.     Rabbit,  intravenous. 

Sarcina  tetragena.     Guinea  pigs  and  white  mice,  subcutaneous. 

Bacterium  anthracis.     Guinea  pigs  or  rabbits,  subcutaneous. 

Bacterium  cholerae.     Rabbits  and  pigeons,  subcutaneous. 

Bacterium  pneumoniae.     Rabbits  and  mice,  subcutaneous. 

Bacterium  pneumoniae.  Rabbits  and  mice,  subcutaneous  with 
sputum. 

Bacterium  pneumonicum.     Mice  and  young  rats,  intraperitoneal. 

Bacterium  tuberculosis.  Guinea  pigs,  rabbits  and  field  mice, 
subcutaneous  or  intraperitoneal. 


1  For  small  animals  a  muffle  furnace  does  very  well. 


306  MEDICAL  BACTERIOLOGY 

Bacterium  mallei.    Male  guinea  pigs,  intraperitoneal. 

Bacterium  diphtheriae.  Guinea  pigs,  rabbits  and  fowl,  subcuta- 
neous and  intratracheal. 

Bacillus  pestis.  Rats,  mice,  guinea  pigs  and  rabbits,  subcuta- 
neous. 

Bacillus  Salmonii.     Rabbits  and  mice,  subcutaneous. 

Bacillus  tetani.     Guinea  pig,  subcutaneous. 

Bacillus  tetani.     White  rat  with  garden  earth. 

Bacillus  Welchii.  Rabbit,  intravenously,  and  then  kill  in  3 
minutes.  See  p.  352. 

Bacillus  Welchii.     Guinea  pig,  subcutaneous. 

Microspira  Metschnikovi.    Pigeons,  subcutaneous. 


MEDICAL  BACTERIOLOGY 


307 


MEDIASTINAL 

AND  BRONCHIAL" 

GLANDS 

LUNGS -^ 
LEFT  LAID   """" 
ON  RIGHT 


BLADDER  *' 


SUBMAXILLARY 
GLANDS 


DEEP  CERVICAL 
GLANDS 

AXILLARY  AND 

SUBSCAPULAR 

GLANDS 

LIVER 


--ADRENAL  GLANDS 
— —  SPLEEN 


RETRO  PERITONEAL 
GLANDS 


SUPERFICIAL 
INGUINAL 
GLANDS 


POPLITEAL  GLANDS 


"-HIND  GUT 


FIG.  37.    Diagram  showing  method  of  making  autopsy  on  guinea  pig;  ana  also  the 
Qost  important  glands  (adapted  from  Delepine  &  Curtis). 


308  MEDICAL  BACTERIOLOGY 

BLANK  FOR  ANIMAL   EXPERIMENTS 

Animal No Sex Age Weight 

Date o'clock    .  , M. 

Inoculated    with    

How  inoculated   

Symptoms  produced : 


Died    (or  killed) o'clock M. 

Autopsy    made o'clock M. 

Autopsy  findings  : 


MEDICAL  BACTERIOLOGY  309 

Bacteriological  examination : 


Histological  Examination : 


Organs  preserved    

Museum    No..  Slide    Nos. 


310 


MEDICAL  BACTERIOLOGY 


EXERCISE  98.     PREPARATION  OF  TISSUE  FOR  EXAMINATION. 

Portions  of  the  diseased  tissue,  removed  at  autopsy,  should  be 
cut  into  cubes  having  edges  about  5  mm.  long  and  treated  as  follows : 

1).  FIXING.  Use  15  or  20  times  their  volume  of  95%  alcohol 
for  24  hours.  The  specimens  should  be  placed  on  cotton  to  keep 
them  near  the  top  and  the  alcohol  changed  after  3  or  4  hours.  If 
they  are  not  to  be  sectioned  immediately  carry  to  80%  alcohol. 

Where  larger  sections  are  desired  they  should  be  left  a  longer 
time  in  the  alcohol. 


2).  PREPARATION  FOR  SECTIONING. 


A. 

Paraffin  Method. 

I 
a.  Absolute  alcohol  6-24  hours. 

I 
6.  Xylene  6-24  hours. 

I 

c.  Paraffin    melting    at    50°    C. 
and  kept  in  an  oven  or  water-bath 
at   a   temperature   a    few   degrees 
above    the    melting    point   of    the 
paraffin  3-12  hours. 

I 

d.  Embed.     Pour     melted     par- 
affin   into   a   paper   box   or   other 
suitable  receptacle  and  with  warm 
forceps  arrange  block  of  tissue  in 
proper  position   and   cool   rapidly 
by  plunging  into  cold  water. 

I 


B. 


Celloidin  Method 

a.  Mixture  of  ether  and 
absolute      alcohol       (equal 
parts)   24  hours. 
I 

6.  Thin  celloidin  (about 
6%)  24  hours  to  several 
weeks. 

I 

c.  Thick  celloidin  (about 
12%)    24   hours   to   several 
weeks. 

d.  Remove   block    of   tis- 
sue to  a  piece  of  wood  fiber 
covered     with     thick     cel- 
loidin.   orient,    dry    a    few 
minutes  in  air,   then  place 
in    80%    alcohol    for    6-24 
hours. 


C. 
Freezing  Method. 

I 

a.  Place  in  1% 
formalin  2  hours. 

I 

7).  Place  tissue  on 
plate  of  freezing  mi- 
crotome in  water,  or, 
better,  first  soak  tis- 
sue in  a  syrupy  so- 
lution of  gum  arable 
and  moisten  plate 
with  same  before 
freezing. 

I 


3).  SECTIONING.     Cut  sections  from  10-12  ^  thick. 

4).  MANIPULATION  OF  SECTIONS. 

a.  Celloidin  sections  can  be  preserved  in  80%  alcohol  and  are  best 
stained  by  placing  the  sections  first  in  water  and  then  in  the  stain. 
The  various  reagents  are  best  used  in  watch  glasses  and  the  sections 
transferred  from  one  to  the  other  by  means  of  a  section  lifter. 

5.  Paraffin  sections  should  be  fixed  to  the  slide  or  cover-glass  as 
follows:  A  water-bath  is  heated  up  to  a  few  degrees  below  the 
melting  point  of  the  paraffin,  the  sections  are  placed  on  the  water 
where  they  will  straighten  out  and  are  then  transferred  to  the  slide, 
or,  more  conveniently  to  the  cover-glass,  by  simply  dipping  the  same 
into  the  water  and  drawing  up  the  section  by  means  of  the  fine 
point  of  'a  pair  of  forceps,  or  a  needle,  draining  off  the  water  and 
drying  the  section  in  an  incubator  for  a  few  hours.  The  sections  are 
more  secure  if  the  cover-glasses  are  first  smeared  with  a  thin  coat  of 


312  MEDICAL  BACTERIOLOGY 

egg  albumin.  When  the  sections  are  once  fixed  to  the  cover  the 
staining  can  be  carried  on  in  the  forceps  as  with  ordinary  cover- 
glass  preparations.  Before  staining,  however,  the  paraffin  must  be 
removed;  this  is  done  with  xylene,  and  this  in  turn  with  absolute 
alcohol. 

REFERENCES.     A.  182 ;  M.  &  W.  204-239 ;  N.  531. 

EXERCISE  99.     STAINING  SECTIONS. 

GENERAL  HISTOLOGICAL  METHOD. 

Hematoxylin  and  Eosin. 

a.  Transfer  sections  from  alcohol  to  distilled  water. 
&.  Stain  in  alum-hematoxylin  5  minutes.     The  stain  may  be 
prepared  as  follows  (Boehmer)  : 

1.  Hematoxylin  crystals, -     -       1  gram. 

Absolute  alcohol, 10  cc. 

2.  Alum,        ----- 20  grams. 

Distilled  water,  -  200  cc. 

Cover  the  solutions  and  allow  them  to  stand  over  night.  The 
next  day  mix  them  and  allow  the  mixture  to  stand  for  one  week  in  a 
wide-mouthed  bottle  lightly  plugged  with  cotton.  Then  filter  into  a 
bottle  provided  with  a  good  cork.  The  solution  is  now  ready  for 
use,  but  its  staining  powers  improve  with  age. 

c.  Acid  alcohol  5  to  10  seconds. 

d.  Ammonia  water  (l1/^)  until  sections  are  a  light  blue. 

e.  "Wash  in  water. 

/.  Counter-stain  with  eosin  (-^  to  y2%  in  60%  alcohol)  3  min- 
utes. 

g.  Alcohol,  95%,  two  or  three  changes  to  dehydrate  and  remove 
excess  of  counter-stain. 

h.  Clear  in  oil  of  origanum,  or  in  Dunham's  mixture  (white  oil 
of  thyme  4  parts,  oil  of  cloves  1  part). 

i.  Balsam. 

GENERAL  BACTERIOLOGICAL  METHODS. 

A.  Loeffler's  Universal  Method. 

a.  Take  sections  out  of  alcohol  and  place  in  Loeffler's  methylen 

blue  for  5  to  30  minutes. 
I.  Decolorize  in  acetic  acid  (0.  1%)  10  to  20  seconds. 

c.  Dehydrate  in  absolute  alcohol,  two  or  three  changes,  a  few 

seconds. 

d.  Clear  in  xylene. 


314  MEDICAL  BACTERIOLOGY 

e.  Mount  in  balsam. 
B.  Weigert' s  Method. 

a.  Lithium  carmine  (carmine  3  gms.,  saturated  aqueous  solution 
of  carbonate  of  lithium,  100  cc.,  a  crystal  of  thymol,  filt- 
ered), 5  minutes. 
Z>.  Acid  alcohol,  15  seconds. 

c.  Wash  in  water. 

d.  Transfer  to  slide  and  blot. 

e.  Ehrlich  's  anilin  water  gentian  violet  3  minutes. 
/.  Blot. 

g.  Place  in  potassium  iodide  and  iodine  solution  (iodine  1  part, 

potassium  iodide  2  parts,  water  100  parts)  2  minutes. 
h.  Blot. 
i.  Decolorize  in  a  mixture  of  anilin  oil  2  parts  and  xylene  1  part, 

2  to  5  minutes. 
j.  Blot. 
Jc.  Mount  in  balsam. 

This  stain  can  only  be  used  with  those  organisms  which  take  the 
Gram  stain,  namely :  Str.  erysipelatos,  M.  pyogenes  var.  albus,  M. 
pyogenes  var.  aureus,  Sar.  tetragena,  Bact  anthracis,  Bact.  pneu- 
moniae,  Bact.  rhusiopathiae,  Bact'  tuberculosis,  Bact.  leprae,  Bact. 
diphtheriae,  Ps.  aeruginosa,  Bact.  Welchii,  B.  Feseri,  B.  oedematis, 
B.  tetani  and  Streptothrix  bovis. 

SPECIAL  BACTERIOLOGICAL  METHODS. 

Particular  organisms  may  be  stained  as  follows : 

Pus  micrococci.     Loeffler  's  or  Weigert 's  method. 

Micrococcus  gonorrhoeae.  Loeffler 's  method  gives  the  best  re- 
sults. 

Sarcina  tetragena.     Loeffler 's  or  Weigert 's  method. 

Bacterium  anthracis.     Loeffler 's  or  Weigert 's  method. 

Bacterium  pneumoniae  (Pneumococcus).     Weigert 's  method. 

Bacterium  pneumonicum  (Friedlander's  bacillus).  The  follow- 
ing method  is  recommended  for  staining  the  capsules  in  sections 
(M.  &  W.): 

a.  Stain  for  24  hours  in  the  incubator  in  the  following  solution : 
Saturated  alcoholic  solution  of  gentian  violet     -       50  cc. 
Distilled  water    ------------     100  cc. 

Glacial  acetic  acid  -  ........  10  cc. 

6.  Wash  out  in  \%  solution  of  acetic  acid. 

c.  Alcohol. 


316  MEDICAL  BACTERIOLOGY 

d.  Xylene. 

e.  Canada  balsam. 

Bacterium  cholerae  (chicken  cholera).     Loeffler's  method. 
Bacterium  tuberculosis. 

a.  Weigert's  method  (staining  with  anilin  oil  gentian  violet  24 
hours  at  room  temperature,  or  2  to  3  hours  at  40°  C.). 

~b.  Ziehl-Neelsen 's  Method. 

1.  Stain  with  carbol-fuchsin  (12  to  24  hrs.  room  temper- 
ature, 1  to  3  hrs.  40°  C.). 

2.  Decolorize  with  nitric  acid  (10%)  a  few  seconds,  and 
then  with  alcohol  (60  to  90%)  until  color  is  nearly  all  extracted. 

3.  Counter-stain  with  methylen  blue. 

4.  Dehydrate  with  absolute  alcohol  (a  few  seconds). 

5.  Clear  with  clove  oil. 

6.  Xylene  (and  examine). 

7.  Mount  in  balsam. 
Bacterium  leprae. 

This  organism  is  stained  with  the  tubercle  stain,  unless  the  sections 
have  been  kept  in  alcohol  for  some  time,  in  which  case  Weigert  's 
method  can  be  employed.     To  differentiate  this  organism  from 
B.  tuberculosis,  stain  as  follows : 
a.  An  aqueous  solution  of  fuchsin  6  to  7  minutes, 
fc.  Acid  alcohol  (nitric  acid  1,  alcohol  10)  14  minute. 

c.  Wash  in  water. 

d.  Counter-stain  in  a  saturated  aqueous  solution  of  methylen 

blue. 

e.  Alcohol. 
/.  Xylene. 
g.  Balsam. 

The  bacteria  of  leprosy  stain  readily  by  this  method,  tuber- 
cle bacteria  do  not. 
Bacterium  mallei. 

Slow  Method. 

a.  Stain  in  Loeffler  's  methylen  blue  6  to  8  hours. 

&.  Wash  in  distilled  water. 

c.  Tannic  acid  solution  (10%)  4  to  5  hours. 

d.  Wash  thoroughly  in  water. 

e.  Dehydrate  in  absolute  alcohol. 
/.  Clear  in  xylene  and  mount. 

Quick  method. 
a.  Stain  in  carbol-methylen  blue  10  to  30  seconds. 


318  MEDICAL  BACTERIOLOGY 

6.  Wash  in  distilled  water. 

c.  Tannic  acid  solution  (10%)  y2  to  1  minute. 

d.  Counter-stain  with  a  weak  solution  of  eosin  until  sections  are 

red. 

e.  Wash  in  water  until  pink. 

/.  Dehydrate  in  absolute  alcohol. 

g.  Clear  in  xylene  and  mount. 

Bacterium  diphtheriae.     Loeffler's  or  better  Weigert's  method. 
Bacillus  typhosus. 

a.  Loeffler's  methylen  blue  or  carbol-fuchsin  15  min.  to  24  hrs. 

b.  Wash  slightly  in  distilled  water. 

c.  Place  in  30%  solution  of  tannic  acid  for  10  to  60  min. 

d.  Dehydrate  rapidly  in  alcohol. 

e.  Clear  in  xylene. 
/.  Examine. 

g.  Mount  in  balsam. 

Such  sections  examined  under  a  low  power  will  be  found  to  con- 
tain heavily  stained  masses,  which  under  a  high  power  prove  to  be 
clumps  of  bacilli.     Not  infrequently  the  bacilli  are  difficult  to  detect 
in  tissue  from  typhoid  cadavers. 
Bacillus  Salmonii  (hog  cholera).     Loeffler's  method. 
Bacterium  WelcJiii  ( gas  bacillus) .    Weigert  's  and  Loeffler  's  methods. 
Bacillus  Feseri  (symptomatic  anthrax).     Use  Pfeiffer's  stain: 

a.  Dilute  carbol-fuchsin  %  hour. 

&.  Absolute  alcohol  slightly  acidulated  with  acetic  acid  until 
section  is  a  reddish  violet  tint. 

c.  Xylene  and  examine. 

d.  Mount  in  balsam. 

Bacillus  oedematis  (malignant  oedema).     Pfeiffer's  stain. 
Streptothrix  bovis  (actinomyces). 

a.  Ziehl's  carbol-fuchsin,  10  minutes. 

6.  Wash  in  distilled  water. 

c.  Picric  acid  (cons.  ale.  solution). 

d.  Wcsh  in  distilled  water. 

e.  Wash  in  alcohol  (50%). 

/.  Dehydrate  in  absolute  alcohol. 

g.  Clear  in  xylene. 

h.  Balsam. 

Tissue  stained  yellow,  rays  red. 

REFERENCES.     M.  &  W.  239-286 ;  N.  537. 


320 


MEDICAL  BACTERIOLOGY 


CHAPTER  IX 


BACTERIOLOGICAL  DIAGNOSIS 


EXERCISE  100.     EXAMINATION  OF  BUCCAL  SECRETION. 

DEFINITION.  The  secretion  of  the  mouth,  or  saliva,  is  a  mixed 
product  derived  in  part  from  the  mucous  glands  within  the  mouth, 
and  also  from  the  parotid,  submaxillary  and  sublingual  glands.  In 
disease  the  normal  character  of  the  different  parts  may  varjr,  or 
there  may  be  various  exudates  and  growths  present. 

COLLECTION.  Material  for  bacteriological  examination  is  best 
obtained  by  means  of  a  sterile  probang  or  by  forceps.  This  material 
may  be  examined  directly  by  means  of  cover-glass  preparations  or 
by  means  of  cultures. 

a.  Method  of  Preparing  Outfit.  Wind  a  small  piece  of  absorbent 
cotton  on  the  end  of  a  wire  (about  1  mm.  in  diameter  and  14  cm. 
long).  Thrust  the  other  end  of  the  wire  through 
the  cotton  plug  of  a  test-tube  or  fasten  in  a  cork  and 
sterilize  at  150°  C.  for  1  hour.  This  with  a  tube  of 
nutrient  medium  (usually  Loeffler's  Blood  serum)  is 
placed  in  a  box  for  transportation.  Fig.  38. 

&.  Method  of  Using  Outfit,  The  patient  is  placed 
in  a  good  light  and  the  probang  gently  but  firmly 
rubbed  over  the  suspected  area  of  the  throat  and  then 
drawn  gently  over  the  surface  of  the  medium,  both  Jj 
tubes  securely  stoppered  and  the  outfit  sent  to  the 
laboratory. 


Tube  1  is  a  ster- 
_  _.  _,  ile  swab;  2  is  a 

ORGANISMS  COMMONLY  FOUND.  blood  serum 

slope. 

Bacterium  diphtheriae. 

The  presence  of  this  germ  in  the  mouth  usually  results  in  the 
formation  of  a  pseudo-membrane,  a  portion  of  which  is  to  be  removed 
with  a  pair  of  forceps,  or  by  means  of  the  outfit  described  above.  It 
should  be  examined  directly  for  the  diphtheria  bacillus  by  smearing 
on  a  cover-glass  and  staining  by  following  methods : 


322  MEDICAL  BACTERIOLOGY 

a.  Loeffler  's  methylen  blue,  or  Roux  stain.1 

&.  Gram's  stain. 

c.  Neisser's  stain:  a.  1  gram  methylen  blue  dissolved  in  20  cc. 
of  alcohol  (96%),  is  added  to  950  cc.  of  distilled  water  and  50  cc. 
of  glacial  acetic  acid;  &.  2  grams  of  bismarck  brown  dissolved  in  a 
Ijter  of  distilled  water.  Films  are  stained  in  a.  5  to  8  seconds,  washed 
in  water,  stained  in  Z>.  3  to  5  seconds,  dried  and  mounted.  The 
Crouch2  stain  may  be  similarly  employed. 

Usually,  however,  mere  microscopical  examination  is  not  suffi- 
cient, and  culture  methods  must  be  employed.  In  fact  this  method 
ought  always  to  be  used.  In  this  case  make  smears  on  Loeffler 's 
blood  serum  and  incubate  them  at  36  to  38°  C.  for  12  to  24  hours  and 
then  examine  the  growth  in  cover-glass  preparations.  The  diph- 
theria organism  if  present,  should  show : 

a.  Characteristic  appearance  with  Loeffler 's  methylen  blue. 

ft.  Positive  Neisser  stain. 

c.  Positive  Gram  stain. 

Occasionally  micro-organisms  (pseudo-diphtheria  bacilli  among 
others)  are  met  with  that  very  closely  resemble  the  Klebs-Loeffler 
bacillus  and  render  a  positive  diagnosis  doubtful.  In  such  cases 
attention  to  following  table  will  be  helpful : 


xBoux  stain— Solution  A:  Dahlia  1,  alcohol  10,  and  distilled  water  90 
parts;  Solution  B:  Methyl  green  1,  alcohol  10,  and  distilled  water  90  parts; 
mix  2  parts  of  A  with  1  of  B. 

8  Crouch  stain — Aqueous  solution  of  dahlia  (1%)  1  part,  aqueous  solution 
of  methyl  green  (1%)  5  parts,  and  distilled  water  4  parts. 


324 


MEDICAL  BACTERIOLOGY 


B.  diphtheriae 

B.  pseudo  diphtheriticum 

1)   Form 

Slender,    and    of    same   di- 

Thicker    at     center     than 

ameter  throughout 

ends,     plumper,     shorter 

and  less  variable  than  B. 

diphtherias 

2)    Size 

Average  1.2  to  2  p 

Averaging  1  to  1.6  /x 

3)    Threads 

Not  formed 

Not  formed 

4)    Grouping 

Parallel   grouping  more  or 

Parallel   but  lie  closer  to- 

less characteristic  but  do 

gether 

not  touch 

5)   Involution  forms 

Common 

Rare 

6)    Motility 

Immotile 

Immotile 

7)    Stains 

a.  Loeffler's    methylen 

Stains  readily  giving  band- 

Stains more  regularly 

blue 

ed  or  polar  stain 

Polar  stain  rare 

6.  Gram. 

Positive 

Positive 

c.  Neisser 

Characteristic     stain     with 

Not  under  24  hours 

very  young  cultures,  six 

hours 

8)    Spores 

Absent 

Absent 

9)   Alkaline  potato 

Growth  almost  invisible 

Visible  and   cream   colored 

10)    Sugar   agar   and   gela- 

in 2  days 

tin  stab  cultures 

Full  length  of  stab 

Only  at  upper  part 

11)    Neutral  litmus  milk 

Acid  reaction 

Alkaline  reaction 

12)    Dextrose  bouillon 

Acid  reaction 

Alkaline  reaction 

13)   Anaerobic    cultures    in 

H. 

Grows  well 

No  growth 

14)    Nitroso-indol    reaction 

After  7  days 

After  21  days 

15)    Inoculation    experi- 

ments    (Guinea    pig 

subcutaneous) 

Death  36-48  hours 

Non-pathogenic 

Pus  Micrococci.  (Str.  erysipelatos,  M.  pyogenes  var.  aureus  and 
albus,  Sar.  tetragena.) 

a.  Stained  cover-glass  preparations  are  to  be  examined,  and  if 
micrococci  are  found  make : 

6.  Smear  cultures,  or  better,  agar  plate  cultures  and  work  up  the 
colonies  as  they  appear. 

Monilia  Candida  (Organism  of  Trush). 

The  material  is  collected  by  removing  a  portion  of  the  patches  or 
membrane  and  examining  it : 

a.  Under  the  microscope  in  a  drop  of  glycerine. 

~b.  Cover-glass  preparations  stained  with  carbol-fuchsin  or  Gram's 
method. 

c.  By  means  of  smear  cultures  on  agar  or  blood  serum,  the  result- 
ing growth  being  examined  either  in  glycerine  mounts,  or  stained 
cover-glass  preparations. 

KEFERENCES.  Em.  43  ;  v.  J.  95 ;  Si.  122.  See  also  various  texts 
under  special  organism. 


326  MEDICAL  BACTERIOLOGY 

EXERCISE  101.— EXAMINATION  OF  SPUTUM. 

DEFINITION.  By  this  term  is  meant  all  of  the  material  derived 
from  the  air  passages  by  the  act  of  coughing  or  hawking. 

METHOD  OF  COLLECTION.  For  diagnostic  purposes  it  is  best  col- 
lected in  a  salt-mouthed  bottle  (about  2  oz.  capacity)  which  has  been 
sterilized.  The  morning  sputum  is  best,  and,  before  being  collected, 
the  mouth  should  be  rinsed  out  with  water. 

ORGANISMS  MOST  COMMONLY  FOUND. 

Bacterium  tuberculosis.  Place  the  sputum  in  a  Petri  dish  over  a 
black  surface  and  select  one  of  the  little  cheesy  masses,  if  these  be 
present,  and  smear  it  on  a  cover-glass.  Where  these  particles  are 
not  present  a  loop  or  two  of  the  thick  portion  is  used.  The  cover- 
glass  preparations  are  to  be  stained  by  one  of  the  following  methods : 

a.  Gabbett,  see  Part  1,  p.  62. 

&.  Ziehl-Neelsen : 

1.  Carbol-fuchsin  ten  times  through  the  flame  (5  to  10  min.). 

2.  Nitric  acid  (30%)  momentarily.1 

3.  Water. 

4.  Alcohol   (60%)    until  red  color  disappears.     It  may  be 
necessary  to  immerse  preparation  in  acid  a  second  time,  but  care 
must  be  exercised  to  prevent  extraction  of  dye  from  tubercle  bac- 
terium. 

5.  Loeffler's  methylen  blue,  1  minute. 

6.  Mount  and  examine. 

While  the  tubercle  bacteria  may  be  detected  when  present  in 
considerable  numbers  with  a  1-6-inch  objective,  when  there  are  few 
present,  a  ^-inch  oil  immersion  will  be  necessary,  and  this  ought  to 
be  used  to  search  all  slides  where  the  tubercle  germ  has  not  been 
found  with  a  lower  power.  A  mechanical  stage  is  a  great  conven- 
ience in  a  systematic  search. 

At  least  two  preparations  should  be  stained  and  thoroughly  exam- 
ined before  a  negative  result  is  pronounced. 

The  viscosity  of  sputa  may  be  overcome  and  the  bacteria  con- 
centrated, where  the  number  is  very  small,  by  1)  Ribbert's  method 
which  consists  in  the  addition  of  a  2%  solution  of  caustic  potash  and 
boiling.  This  dissolves  the  mucus,  and  the  bacteria  are  then  de- 
posited with  the  sediment.  This  sediment  can  be  obtained  by  allow- 


1  Eavenel  recommends  use  of  5%  nitric  acid  in  80%  alcohol,  claiming  that 
there  is  no  danger  of  decolorizing  the  tubercle  bacilllus  no  matter  how  long 
the  contact. 


328  MEDICAL  BACTERIOLOGY 

ing  the  mixture  to  stand  in  a  conical  glass  vessel  or,  more  quickly,  by 
the  use  of  a  centrifuge.     2 )  Hammond 's  method : 

a.  Add  5%  of  crystallized  carbolic  acid  (in  the  case  of  sputum 
add  5  times  its  bulk  of  a  5%  solution  of  carbolic  acid). 

&.  Place  15  cc.  in  the  tubes  of  a  centrifuge  and  whirl  for  15 
minutes. 

c.  Pour  off  supernatant  fluid  and  treat  precipitate  with  3  cc.  of  a 
6%  KOH  solution.     Mix  thoroughly  and  allow  to  stand  2  minutes. 

d.  Fill  to  15  cc.  mark  with  distilled  water  and  whirl  20  minutes. 

e.  Make  cover-glass  preparation  of  sediment  (or  purify  same  by 
repeated  washings  and  centrif legalizations  with  distilled  water). 

A  centrifugal  machine  should  be  able  to  make  at  least  2,500 
revolutions  per  minute.  This  speed  ought  to  be  maintained  for  15 
minutes.  Sputum  may  be  preserved  by  addition  of  a  small  quantity 
of  carbolic  acid  (5%). 

Negative  results  are  of  positive  diagnostic  value  only  when  re- 
peated examinations  are  made  of  different  samples  taken  at  different 
times. 

EEFERENCES.     Em.  75. 

Bacterium  influenzae.  This  micro-organism  is  frequently  pres- 
ent in  enormous  numbers  (100  or  more)  and  sometimes  in  almost 
pure  cultures  in  the  greenish  purulent  masses  in  the  sputum/  It 
stains  readily  with  the  ordinary  dyes,  and  when  lightly  stained 
presents  the  bipolar  stain.  Carbol-fuchsin  diluted  10  times  with 
distilled  water  is  one  of  the  best  stains.  Gram 's  stain  is  negative. 

Sputum  from  suspected  cases  should  be  collected  either  by  means 
of  a  probang,  or  in  a  bottle,  and  examined : 

1)  Microscopically   by   staining,   with   a   weak   carbol-fuchsin, 
smears  from  the  purulent  masses.     If  a  very  small  bacillus  is  in 
large  clumps,  which  fails  to  retain  stain  by  Gram's  method,  the 
evidence  is  strong  that  it  is  the  influenza  bacillus;  the  diagnosis 
should  be  confirmed,  however,  by 

2)  Cultures  on  blood  agar. 

Animal  inoculations  are  without  effect. 
REFERENCES.     Em.  64. 

Bacterium  pneumoniae  (pneumococcus). 

The  sputum  of  patients  suffering  from  pneumonia  is  usually  of 


330  MEDICAL  BACTERIOLOGY 

a  rusty  color,  due  to  presence  of  blood  ( rusty  sputum ) .  The  ' '  pneu- 
mococcus ' '  is  readily  seen  in  such  material  when  stained  by  Gram 's 
method  or  with  carbol-fuchsin  and  momentarily  washed  with  alco- 
hol, as  lancet-shaped  organisms  with  outer  ends  pointed  and  sur- 
rounded by  a  clear  area— the  capsule.  The  capsule  can  be  easily 
stained  by  Welch 's  method.  ( See  27. ) 

This  organism  is  also  frequently  found  in  the  sputum  of  healthy 
persons  and  small  numbers  may  be  detected  by  means  of  animal 
inoculation.  The  rabbit  or  mouse  is  most  susceptible  and  should  be 
inoculated  subcutaneously.  As  a  result  of  infection  with  this  organ- 
ism the  animal  dies  quickly  with  a  typical  septicemia,  the  micro- 
organisms being  found  in  great  numbers  in  the  blood  current. 

Bacillus  pestis.  This  micro-organism  is  frequently  found  in  the 
sputum,  especially  in  the  pneumonic  form  of  the  disease— for  meth- 
ods of  detection  see  105. 

Streptothrix  bovis  (actinomyces).  This  organism  has  been  occa- 
sionally found  in  sputum  and  in  such  cases  the  peculiar  morphology 
of  the  colonies  is  well  brought  out  by  Gram 's  method.  See  105. 

REFERENCES,  v.  J.  114;  Si.  245.  See  also  various  texts  under 
particular  organisms. 


EXERCISE  102.  EXAMINATION  OF  BLOOD. 

COLLECTION.  For  serum  test  (Widal  reaction)  the  blood  may  be 
collected  and  dried  (see  below),  but  in  other  cases  where  cultures  are 
to  be  made,  the  blood  must  be  collected  aseptically  in  sterile  recepta- 
cles and  hermetically  sealed.  For  this  purpose  Sternberg's  bulb  is 
excellent.  The  skin  should  first  be  sterilized  by  use  of  corrosive  sub- 
limate or  carbolic  acid  followed  with  alcohol. 

It  is  usually  well  in  any  case  to  make  cover-glass  smears  at  the 
bed-side  for  microscopical  examination.  These  are  best  made  as 
follows:  Place  a  drop  of  blood  about  the  size  of  a  pin  head  on  a 
perfectly  clean  cover-glass  and  then  place  a  second  cover-glass  on 
this ;  this  flattens  the  drop  of  blood  out  into  a  thin  film.  Immedi- 
ately and  before  coagulation  can  take  place  the  two  are  drawn  apart 
horizontally  and  the  films  allowed  to  dry.  ( Cabot. ) 

Bacterium  anthracis.  In  case  of  animals  dead  of  suspected  an- 
thrax, blood  or  portion  of  spleen  should  be  removed  with  least  pos- 


332  MEDICAL  BACTERIOLOGY 

sible  danger  from  infection  or  distribution  of  bacilli  and  studied  as 
follows : 

a.  Microscopical  examinations  of  blood  or  of  the  spleen  pulp  of 
animals  show  (when  stained  with  Loeffler's  methylen  blue)  large 
bacteria  in  chains  (5  or  6  segments)  presenting  the  bamboo  appear- 
ance. 

~b.  In  hanging  drop  preparation  large,  homogeneous,  immotile 
bacilli. 

c.  Agar  plate  cultures  should  also  be  made,  and,  from  the  separate 
colonies,  subcultures ;  the  gelatin  stab  being  especially  characteristic. 

d.  In  important  cases  (as  in  man)  guinea  pigs,  or  white  mice, 
should  be  inoculated,  and,  in  case  of  death,  organism  isolated  and 
identified. 

Spirockaeta  Obermeieri  (relapsing  fever).  This  organism  is 
found  in  the  blood  only  during  a  paroxysm.  It  is  a  long  slender 
organism  6  or  7  times  the  diameter  of  a  red  blood  corpuscle  (45/*). 
It  has  a  brisk,  vibratile  movement  in  the  direction  of  its  long  axis, 
and  is  very  sensitive  to  reagents  of  all  kinds.  Even  the  addition  of 
distilled  water  will  cause  it  to  disappear.  Fresh  blood  is  best,  but 
dried  smears  may  be  used  and  stained  with  f uchsin,  or  by  Gunther  's 
method : 

a.  Dried  films  are  treated  with  acetic  acid  (5%)  10  seconds,  this 
is  removed  by  blowing  and  holding  film  over  flask  of  strong  ammonia 
previously  shaken. 

&.  Stained  in  Ehrlich's  gentian  violet. 

c.  Washed  with  water. 

d.  Dried. 

e.  Mounted  in  balsam  or  xylene. 
/.  Examined. 

Pus  Micrococci.  These  are  occasionally  found,  and  for  method 
of  detection  see  105. 

Bacterium  mallei.  Sometimes  found  in  the  blood  of  those  suffer- 
ing with  glanders.  It  may  be  detected  in  the  blood- smears.  For 
special  methods  see  105. 

Bacterium  pneumoniae  (pneumococcus) .  This  germ  is  frequent- 
ly present  in  fatal  cases  24  to  48  hours  before  death.  The  blood 
should  be  drawn  with  a  sterile  hypodermic  syringe  and  about  1  cc.  of 
blood  mixed  with  a  tube  of  melted  agar  at  43°  C.  and  poured  into  a 
Petri  dish.  Characteristic  colonies  appear  in  24  to  48  hours. 


334  MEDICAL  BACTERIOLOGY 

Bacterium  tuberculosis.  In  case  of  miliary  tuberculosis  they 
may  be  very  rarely  found  in  sufficient  numbers  to  be  detected  by 
staining  methods,  see  sputum  101. 

Bacterium  influenzae.  Canon  claims  to  have  stained  and  culti- 
vated this  organ  in  blood,  but  this  needs  confirmation. 

Bacillus  coli.  This  organism  may  be  found  in  the  blood.  For 
methods  of  isolation  and  identification  see  f  eces  103. 

Bacillus  pestis.     This  germ  occurs  in  the  blood,  in  certain  cases 
at  least.     Considerable  skill  in  detecting  it  is  required— due  to  its 
variable  appearance.     Broth  tubes  should  be  infected  and  animals 
inoculated. 
Bacillus  Salmonii  (hog  cholera). 

a.  Make  agar  plate  and  streak  cultures  from  spleen  of  dead  ani- 

mal, and  work  up  the  colonies  as  they  appear. 

b.  Widal  Reaction  (for  technique  see  below  under  B.  typhosus). 
Plasmodium  malariae. 

a.  Examination  of  fresh  blood.     A  droplet  of  blood  from  finger, 

or  from  lobe  of  ear,  is  placed  on  a  glass  slide,  covered  with 
a  cover-glass  and  then  the  cover-glass  is  ringed  with  vaselin. 
Examination  should  be  made  with  a  -fa  in.  oil  immersion. 

b.  Stained.     Prepare  films  as  directed   above   and  stain   with 
methylen  blue  and  eosin,  or  treat  films  with  a  very  weak 
acetic  acid,  2  or  3  drops  to  30  cc.  of  water ;  to  remove  hemoglo- 
bin, wash  with  water  and  stain  with  following  solution  for  y2 
minute : 

Borax 5.0  parts. 

Methylen  blue 0.5  parts. 

Water 100  parts. 

Wash,  dry  and  mount  in  balsam  (Manson). 
REFERENCES,     v.  J.  45  ;  Si.  100.     See  also  texts  under  particular 
organisms. 

WIDAL  REACTION.  Dried  blood  method.  This  method  is  espe- 
cially valuable  where  patient  is  some  distance  from  the  laboratory. 
Collect  the  blood  as  follows:  "Wash  with  boiled  water  the  part 
from  which  the  blood  is  to  be  obtained  (lobe  of  ear,  end  of  finger,  or 
toe  in  infant).  Prick  deeply  the  skin  with  a  needle, "  Remove  two 
or  three  large  drops  of  blood  on  a  clean  glass  slide,  aluminum  foil, 
piece  of  isinglass  or  letter  paper.  Allow  the  blood  to  dry.  Then 
place  in  an  envelope  and  send  to  laboratory  and  test  as  follows : 


$36  MEDICAL  BACTERIOLOGY 

a.  Make  a  hanging  drop  preparation  from  a  24  to  72-hour  old 
agar,  or  bouillon,  culture  of  Bacillus  typhosus. 

5.  If  the  bacilli  be  actively  motile,  remove  the  cover-glass,  add 
to  the  culture  a  small  drop  of  a  solution  of  typhoid  blood  (diluted 
from  10-50  times),  return  the  cover  glass  to  the  slide  and  seal  well 
with  vaselin. 

c.  Examine  with  a  high  dry  power  (1-6  in  obj.)  rather  than  with 
the  oil  immersion. 

The  dilution  is  made  in  the  following  way :  Nine  drops  of  sterile 
water  are  placed  around  the  drop  of  dried  blood.  (The  drops  of 
water  should  be  of  about  the  same  size  as  that  of  the  original  drop  of 
blood.)  The  drops  are  all  mixed  together  and  allowed  to  soak  up 
the  blood  for  about  two  minutes.  In  this  way  an  approximate  dilu- 
tion of  one  to  ten  is  obtained.  One  drop  of  this  is  added  to  the 
hanging-drop  culture.  This  gives  a  dilution  of  one  to  twenty  which 
is  the  one  usually  employed. 

More  exact  dilutions  of  dried  blood  may  be  made  by  weighing  out 
the  blood  and  adding  it  to  a  measured  amount  of  water. 

Where  possible  the  blood  should  be  collected  so  that  the  clear 
serum  may  be  separated  and  used  for  the  test.     This  can  be  done  in 
hospital  work  and  wherever  it  is  possible  to  get  the  blood  to  the 
laboratory  a  few  hours  after  it  is  collected.     For  this  purpose  a  glass 
pipette  is  prepared  by  drawing  out  a  glass  tube,  as  indicated 
in  Fig.  39,  which  represents  the  pipette  natural  size.     The 
skin  is  cleaned  and  the  blood  drawn  as  indicated  above  and 
when  a  large  drop  has  collected  on  the  skin  one  of  the  points 
of  the  pipette  is  introduced  when  the  blood  is  drawn  up  by 
capillary  attraction.     The  bulb  ought  to  be  about  one-half 
filled.     The  pipette  is  then  placed  in  a  horizontal  position 
until  the  blood  has  clotted,  when  it  may  be  taken  to  the 
laboratory.     It  should  then  be  placed  in  the  ice  chest,  still 
in  a  horizontal  position,  for  two  or  three  hours.     The  end 
which  was  used  to  draw  up  the  blood  is  then  scratched  with 
a  file  and  broken  off.     By  holding  the  tube  in  a  vertical 
F  position  the  clear  serum  may  now  be  dropped  from  the 

B. !  oo  a  opposite  end  into  a  glass  or  porcelain  capsule.     The  clear 

plp6trtJG« 

serum  is  then  taken  up  with  a  clean  capillary  pipette  and  a 
drop  placed  in  another  capsule  and  then  after  rinsing  out  the  same 
pipette  is  used  to  add  the  requisite  number  of  drops  of  bouillon  or 
salt  solution  to  make  the  required  dilution.  The  test  is  then  made 
in  exactly  the  same  way  as  described  for  the  dried  blood, 


338  MEDICAL  BACTERIOLOGY 

In  a  typical  reaction  the  motility  is  almost  immediately  affected, 
and  soon  motion  ceases  altogether  while  the  bacilli  collect  in  clumps, 


FIG.  40.    Widal  Reaction.    I.  B,  typhosus  before  adding  typhoid  blood;    II,  A  typical 
reaction. 

i.  e.,  become  "agglutinated."  (Fig.  40.)  The  usual  time  limit  is 
thirty  minutes  when  the  dilution  is  1  to  50. 

REFERENCES,  v.  J.  45 ;  Si.  100.  See  also  texts  under  particular 
organism. 

EXERCISE  103.     EXAMINATION  OF  FECES. 

The  material  expelled  from  the  rectum  and  comprising  the  sub- 
stances from  the  food  and  the  secretions  of  the  alimentary  tract  come 
under  this  head.  The  number  of  micro-organisms  occurring  here  is 
enormous,  and  comprise  a  large  number  of  species  and  among  them 
several  pathogenic  forms  particularly  B.  typhosus,  Msp.  comma, 
Bact.  tuberculosis  and  Amoeba  coli. 

Bacillus  typhosus.  This  organism  occurs  in  the  f eces  in  the  case 
of  typhoid  patients;  but  on  account  of  the  large  number  of  other 
organisms  its  detection  is  very  difficult.  The  following  methods  are 
the  most  serviceable : 

A.  PARIETTI'S  METHOD.  This  method  consists  in  adding  Parietti's 
solution  (carbolic  acid  5  grams,  hydrochloric  acid  4  grams,  and  dis- 
tilled water  100  cc. )  to  bouillon  in  the  following  manner :  A  num- 
ber of  tubes  of  bouillon  have  a  varying  quantity  of  the  above  solu- 
tion added,  e.  g.  1  drop  to  one  tube,  2  to  another,  3  to  another,  and 
so  on.  These  tubes  are  inoculated  with  a  small  quantity  (one  or  two 
loops),  of  the  feces  and  then  placed  in  the  38°  C.  incubator.  Twen- 


340  MEDICAL  BACTERIOLOGY 

ty-four  hours  later  the  tube  containing  the  largest  amount  of  Pari- 
etti's  solution  which  shows  growth  probably  contains  B.  coli  and  B. 
typhosus  if  it  is  present.  The  organisms  may  be  separated  most 
quickly  and  easily  by  the  use  of  the  lactose  litmus  agar  plate.  The 
blue  colonies  should  be  worked  up,  and  especially  tested  for  their 
agglutinating  power  on  typhoid  blood.  Instead  of  the  lactose  litmus 
agar  one  of  the  following  media  may  be  used : 

B.  Hiss'  PLATE  MEDIUM.1     This  contains: 

10  grams  of  agar. 
25  grams  of  gelatin. 
5  grams  of  beef  extract  (Liebig). 
5  grams  of  sodium  chloride. 
10  grams  of  dextrose. 
1000  grams  of  water. 

It  is  made  by  first  dissolving  the  agar,  salt  and  extract  in  the 
water,  then  the  gelatin  is  added  and  dissolved,  the  reaction  changed 
by  use  of  NaOH  and  phenolphthalein  so  that  it  will  contain  not  less 
than  2%  normal  acid,  cleared  with  two  eggs  and  filtered,  dextrose 
added  and  the  medium  tubed  and  sterilized. 

Make  plate  cultures  in  ordinary  way  and  incubate  at  38°  C.  for 
18  hours,  then  examine  the  colonies  microscopically.  The  colonies 
of  B.  typhosus  have  irregular  outgrowths  and  fringing  threads.  The 
colonies  of  B.  coli,  on  the  other  hand,  are  much  larger  and  as  a  rule 
are  darker  in  color  and  do  not  form  threads. 

The  colonies  may  be  further  examined  by  the  use  of  Hiss'  Tube 

Medium. 

5  grams  of  agar-agar. 
80  grams  of  gelatin. 
5  grams  of  beef  extract   (Liebig). 
5  grams  sodium  chloride. 
10  grams  dextrose. 
1000  grams  water. 

Made  as  plate  medium  except  that  it  is  to  contain  1.5%  normal 
acid. 

Within  18  hours  at  38°  C.  the  typhoid  bacilli  produce  a  uniform 
clouding.  The  colon  bacilli  do  not  produce  uniform  clouding  and 
do  produce  gas. 

C.  MEDIUM  OF  MACCONKEY,  as  modified  by  Griinbaum.8 


i  Jour.  Exp.  Med.  1897,  2:  677. 

*  Brit.  Med.  Jour.  1902,  Pt.  1,  p.  1473. 


342  MEDICAL  BACTERIOLOGY 

Twenty  grams  each  of  agar-agar  and  peptone  are  dissolved  in 
one  liter  of  boiling  water,  and  the  whole  made  alkaline  by  adding  4 
cc.  of  a  normal  solution  of  sodium  hydrate  after  having  first  brought 
the  reaction  to  the  neutral  point  of  litmus. 
Then  add,  while  hot, 

Sodium  taurocholate       .----....      5  grams. 

Lactose          10  grams. 

Neutral  Red  (5%  watery  solution) 10  cc 

When  the  solution  is  complete  the  mass  is  filtered  through  cotton, 
tubed,  and  sterilized  in  the  steam  sterilizer  once  for  twenty-five  or 
thirty  minutes. 

D.  MEDIUM  OF  DRIGALSKI  AND  CONRADI.' 
To  two  liters  of -sugar-free  broth  add: 
Peptone  (Witte)  -     -     -     10  grams 

Nutrose        10  grams  ) 

e,    -,.          1,1     -j  -i  A  >  these  may  be  omitted 

Sodium  chloride        -     -     10  grams  [ 

and  dissolve  by  the  aid  of  heat.  The  mixture  is  brought  to  the  boil 
and  sixty  grams  of  agar-agar  added,  and  the  mixture  kept  boiling 
until  the  agar  is  dissolved.  Then  the  reaction  of  the  mass  is  made 
weakly  alkaline  to  litmus  by  the  addition  of  sodium  hydrate  (4% 
sol.)  and  filtered. 

This  being  done,  a  mixture  of  litmus  solution  (6%)  and  lactose 
(c.  p.) 

Litmus  sol.     ------......     260  cc. 

Lactose        30  grams. 

is  added  while  both  solutions  are  hot,  and  the  whole  boiled  gently  for 
five  minutes.  Then  add  a  solution  of  water-free  sodium  carbonate 
(10%)  in  the  proportion  of  4  cc.  (this  may  be  omitted)  followed  by 
20  cc.  of  a  fresh  solution  of  crystal  violet  (Griibler's)— 0.1  gram  in 
100  cc.  water— tubed,  and  sterilized  in  the  steam  sterilizer  for  20 
minutes  on  three  successive  days. 

All  suspected  cultures  should  be  tested  with  typhoid  blood 
(Widal  reaction). 

The  typhoid  organism  may  be  isolated  from  the  stools  during  the 
first  two  weeks  of  the  disease. 

Microspira  comma  (Asiatic  cholera). 

1.  Microscopal    examination    of    "rice-water"    discharges    for 
spirilla  lying  parallel. 

2.  Culture  methods.     Gelatin  or  agar  plates  should  be  made 
from  the  rice-like  flakes ;  other  flakes  should  be  inoculated  into  flasks 

*  Zeit.  f .  Hyg.,  1902,  Heft  ii,  p.  283. 


344  MEDICAL  BACTERIOLOGY 

of  peptone  water  (Dunham's  solution)  and  incubated  at  38°  C. 
The  surface  growth  6-12  hours  later  is  to  be  examined  microscopi- 
cally and  by  means  of  plates.  Then  test  the  peptone  cultures  for 
nitroso-indol  (cholera  red  reaction)  by  the  addition  of  a  few  drops 
of  sulphuric  acid. 

B.  dysenteriae.  This  organism  has  been  isolated  from  the  feces 
of  dysentery  patients  by  numerous  investigators  and  from  children 
suffering  from  summer  diarrhoea  by  Duval  &  Bassett1  and  others. 
The  following  method  is  recommended : 

Agar  plates  are  made  from  the  bloody  mucus  in  the  feces  or 
from  scrapings  of  the  ulcerated  mucosa  of  the  intestines.  Agar 
plates  are  made  and  incubated  at  38°  C.  for  12  hours  and  then  the 
colonies  which  have  appeared  are  marked  with  a  pencil  or  pen  and 
then  the  plate  is  incubated  for  several  hours  longer.  The  colonies 
which  appear  later  are  most  likely  to  be  colonies  of  B.  dysenteriae. 
The  suspected  colonies  are  then  put  into  dextrose  agar  and  only 
those  which  fail  to  produce  gas  are  tested  farther.  The  crucial  test 
is  the  Widal  reaction  which  can  be  made  with  blood  obtained  from 
the  patient  or  cadaver. 

Bacterium  tuberculosis.  This  organism  has  been  found  in  the 
stools  in  cases  of  intestinal  ulcerations,  and  may  come,  in  cases  of 
phthisis,  from  ingested  sputa. 

Ameba  coli. 

a.  A  drop  of  the  mucus  portions  of  stool  is  placed  on  a  glass 
slide,  covered  with  a  cover-glass  and  examined  with  a  magnification 
of  about  500  diameters  (1-6  in  objective).  Examination  should  be 
conducted  on  a  warm  stage  in  order  to  get  ameboid  movements. 

Z>.  Preparations  may  be  stained  with  methylen  blue  and  carmine. 
The  nucleus  is  stained  with  carmine. 

c.  Discharges  may  be  hardened  and  stained  by  Mallory  's  method 
as  follows : 

1.  Fix  tissues  in  alcohol. 

2.  Stain  (paraffin)  sections  in  a  saturated  aqueous  solution  of 
thionin  for  5-20  minutes. 

3.  Wash  in  water. 

4.  Differentiate  in  a  2%  aqueous  solution  of  oxalic  ocid  %-l 

minute. 

5.  Wash  in  water. 


1  Duval  and  Bassett,  Amer.  Med.,  1904,  4:  417. 


346  MEDICAL  BACTERIOLOGY 

6.  Dehydrate  in  alcohol  (95%), 

7.  Clear  in  oil  of  bergamot. 

8.  Wash  with  xylene  and  mount  in  balsam. 
Nuclei  of  Amebae  brownish  red,  other  nuclei  blue. 
REFERENCES,     v.  J.  199 ;  Si.  206.     See  also  texts  under  various 

organisms. 

EXERCISE  104.     EXAMINATION  OF  URINE. 

For  bacterial  examination  urine  should  be  drawn  with  a  sterile 
catheter  into  a  sterile  bottle. 

Bacterium  tuberculosis. 

For  method  of  staining  see  under  Sputum,  101. 

It  is  best  to  centrifuge  the  product  and  care  must  be  taken  to 
differentiate  from  the  smegma  bacterium.  For  this  pur- 
pose stain  cover-glass  smears  as  follows  (Bunge  &  Fran- 
teroth)  : 

a.  Absolute  alcohol,  3  hours. 

b.  Chromic  acid,  15  minutes. 

c.  Stain  in  hot  carbol-fuchsin. 

d.  Decolorize  in  sulphuric  acid  (25%)  2-3  minutes. 

e.  Counter-stain  with  a  saturated  alcoholic  solution  of  methylen 

blue. 

The  smegma  bacillus  is  decolorized  by  this  method. 

The  tubercle  bacterium  in  urine  is  frequently  present  in  clusters 
while  the  smegma  bacterium  occurs  singly.  Injection  of  guinea 
pigs,  smegma  bacillus  is  non-pathogenic. 

The  following  organisms  have  also  been  found  in  the  urine.  For 
methods  of  isolation  see  references. 

Pus  Micrococci.     105. 

Micrococcus  gonorrhoeae.     105. 

Bacillus  typhosus.     103. 

Spirochaeta  Obermeieri  (relapsing  fever).     102. 

REFERENCES,  v.  J.  273;  Si.  500,  and  texts  under  the  various 
organisms. 

EXERCISE  105.     EXAMINATION  OF  TRANSUDATES  AND  EXUDATES. 

The  material  should  be  collected  in  sterile  vessels  under  aseptic 
precautions.  Make  several  cover-glass  preparations  and  stain  one 
with  Loeffler's  methylen  blue  and  the  others  with  gentian  violet  or 
carbol-fuchsin.  Mount  and  examine. 


348  MEDICAL  BACTERIOLOGY 

a.  If  staphylococci  alone  are  present  search  for  the  Pus  Coccus  Group. 
6.  If  streptococci  suspect  Str.  erysipelatos. 

c.  If  diplococci  or  tetracocci. 

1.  Within  the  pus-cells  test  for  M.  gonorrhoeae  or  M.  Weichsel- 

baumii. 

2.  Free  suspect  Sar.  tetragena. 

d.  If  bacilli  any  of  the  following  may  be  searched  for: 

1.  B.  coli.  This  organism  is  likely  to  be  found  especially  in  suppu- 
rative  peritonitis  and  diseases  of  the  urinary  organs.  2.  Bact.  anthracis. 
3.  Bact.  pneumoniae.  4.  Bact.  tuberculosis.  5.  Bact.  leprae.  6.  Bact.  mal- 
lei. 7.  B.  pestis.  8.  Ps.  aeruginosa.  9.  Bact.  Welchii.  10.  B.  oedematis. 
11.  B.  tetani. 

e.  Streptothrix  bovis. 
/.  Ameba  coli. 

Pus  Micrococci.  These  organisms  are  frequently  present  in  .pus 
and  should  be  isolated  and  identified  in  pure  cultures,  as  micro- 
scopical examinations  alone  will  not  suffice. 

Streptococcus  erysipelatos.  This  organism  is  not  infrequently 
present  and  can  be  readily  identified  by  culture  methods. 

Micrococcus  gonorrhoeae.  Pus  should  be  collected  in  a  sterile 
receptacle  or  spread  on  cover-glasses  and  allowed  to  dry.  When 
once  dried  it  should  not  be  wet  or  moistened  again  as  this  would 
destroy  the  pus-cells,  and  hence  the  value  of  the  material  for  diag- 
nosis. 

a.  Simple  stain. 

1.  Loeffler's  methylen  blue  3-5  minutes. 

2.  Wash  in  water. 

3.  Dry,  mount  in  balsam  and  examine  with  -fa  in.  oil  immer- 

sion. 

4.  Look  for  a  biscuit-shaped  diplococcus  within  the  pus  cells, 
fe.  Gram's  method. 

1.  Anilin  oil  gentian  violet  15  minutes. 

2.  Wash  in  water. 

3.  Treat  with  iodine  solution  2  minutes. 

4.  Decolorize  with  alcohol. 

5.  Counter-stain  with  eosin,  y2  minute. 

6.  Wash,  dry  and  mount  in  balsam. 

7.  Examine  with  oil  immersion. 

If  the  gonococci  be  present  they  will  be  stained  brown. 
If  diagnosis  be  of  great  importance  make  cultures  as  follows : 
a.  Make  6  or  more  streak  cultures  on  blood  agar,  or  better,  make 
plates  on  Wertheim's  medium  (p.  158).     Grow  at  38°  C. 


350  MEDICAL  BACTERIOLOGY 

6.  Make  a  set  of  ordinary  agar  plates,  or  streak  cultures,  and 
keep  at  38°  C. 

The  gonococcus  grows  on  the  first  two  media,  but  not  on  the  plain 
agar.  The  gonococeus  is  the  only  organism  that : 

1.  Occurs  in  groups  (cell-colonies)  in  pus-cells. 

2.  Is  decolorized  by  Gram's  method. 

3.  Does  not  grow  on  agar  at  room  or  blood  heat. 
Micrococcus  Weichselbaumii  (M.  intracellularis). 

Pus  may  be  obtained  by  lumbar  puncture  which  is  performed  as 
follows :  The  back  of  the  patient  and  the  operator  's  hands  should 
be  made  sterile.  The  needle  (4  cm.  X  1  mm.  for  children)  should 
be  boiled  10  minutes.  The  patient  should  lie  on  the  right  side,  with 
the  knees  drawn  up  and  the  uppermost  shoulder  so  depressed  as  to 
present  the  spinal  column  to  the  operator.  The  puncture  is  gen- 
erally made  between  the  third  and  fourth  lumbar  vertebrae.  The 
thumb  of  the  left  hand  is  pressed  between  the  spinous  processes,  and 
the  point  of  the  needle  is  entered  about  1  cm.  to  the  right  of  the 
median  line,  and  on  a  level  with  the  thumb  nail,  and  directed  slightly 
upwards  and  inward,  toward  the  median  line.  At  a  depth  of  3  or 
4  cm.  in  children  and  7  or  8  in  adults  the  needle  enters  the  subarach- 
noid  space  and  the  fluid  flows  usually  by  drops.  This  is  allowed  to 
drop  into  an  absolutely  clean  test-tube,  which  has  previously  been 
plugged  and  sterilized.  From  5  to  15  cc.  of  the  fluid  is  a  sufficient 
quantity  for  examination.  Cultures  should  be  made  at  once  on 
blood  agar  and  on  plain  agar  (M.  &  W.  371).  After  standing  some 
hours,  the  sediment  should  be  examined  in  cover-glass  preparations, 
stained  with  Loeffler's  methylen  blue  and  by  Gram's  method. 

Microccus  Weichselbaumii  stains  by  Loeffler's  method  and  ap- 
pears as  a  diplococcus  in  groups  in  the  pus  cells,  is  decolorized  by 
Gram's  method,  and  grows  on  blood-agar,  and  feebly  on  ordinary 
agar  at  38°  C. 

The  following  organisms  are  also  found  occasionally.  For  meth- 
ods of  diagnosis  see  exercises  indicated. 

Bacillus  coli.     103. 

Bacterium  tuberculosis.     101. 

Bacterium  leprae.     For  method  of  staining,  see  99. 

Bacterium  pneumoniae.  Stain  for  capsule.  Cultivate  on  blood- 
agar.  101. 

Bacterium  mallei. 

a.  Widal  reaction.  (If  in  man,  typhoid  and  diphtheria  must  be 
excluded  in  case  of  a  positive  reaction.) 


352  MEDICAL  BACTERIOLOGY 

b.  Examination  of  discharge. 

1.  Microscopical  examination  usually  without  result. 

2.  Cultures,  glycerine  agar  and  potato  from  pus. 

c.  Animal  inoculations,  Straus'  method. 
Bacillus  pestis. 

a.  Make  plate  cultures  from  blood  and  buboes  and  work  up 
colonies. 

b.  Make  subcutaneous  inoculation  into  guinea  pigs  from  bubo, 
and  if  death  ensues  search  for  B.  pestis. 

Pseudomonas  aeruginosa  (B.  pyocyaneus).  Easily  recognized 
by  its  culture  characters. 

Bacterium  Welchii  (gas  bacillus). 

This  germ  is  non-pathogenic  for  rabbits,  but  Welch  and  Flexner 
have  shown  that  if  a  rabbit  be  inoculated  intravenously  with  0.5  to 
1  cc.  of  a  bouillon  culture  and  killed  after  a  lapse  of  5  or  10  minutes. 
and  the  animal  kept  at  18°-20°  C.  for  24  hours  or  at30°-35°  C.  for 
4  to  6  hours,  the  organism  will  multiply  in  the  blood  and  produce 
large  quantities  of  gas  in  the  vessels  and  organs.  This  effect  is 
characteristic. 

Bacillus  oedematis  (B.  malignant  oedema). 

a.  Make  cover-glass  preparations  from  fluid  of  affected  parts. 

b.  Also  make  anaerobic  cultures.     If  material  contains  spores  It 
should  be  heated  to  80°  C.  for  10  minutes  before  it  is  seeded. 

Bacillus  tetani. 

a.  Make  cover-glass  preparations  from  pus  and  search  for  drum- 
stick bacillus. 

b.  Make  dextrose  bouillon  and  agar-plate  cultures  and  develop 
in  hydrogen. 

c.  Inoculate  animals  with  the  discharge,  and  also  with  the  bouil- 
lon culture,  and  watch  for  characteristic  symptoms. 

Streptothrix  bovis  (actinomyces). 

a.  Place  one  of  the  minute  sulphur-yellow  nodules  in  a  drop  of 
glycerine  on  a  glass  slide  and  then  apply  gentle  pressure. 

b.  Even  the  low  powers  of  a  compound  microscope  will  then  show 
something  of  the  clustered  arrangement  which  can  be  more  carefully 
studied  under  a  higher  power. 

c.  Intraperitoneal  inoculation  of  guinea  pig.     One  month  later, 
nodules  on  peritoneum. 

Ameba  coli.     103. 

REFERENCES,  v.  J.  405 ;  Si.  514  and  518.  See  also  texts  under 
the  various  organisms. 


354  MEDICAL  BACTERIOLOGY 

EXERCISE  106.     DIAGNOSIS  OF  RABIES. 

A.  Microscopical  Diagnosis. 

a.  The  head  of  the  animal  is  opened  and  the  brain  removed. 
In  case  the  animal  is  some  distance  from  the  laboratory  it  is  best  to 
cut  off  the  head,  pack  in  ice  and  ship  by  express. 

b.  Thin    pieces   of   the    various   parts    of   the   brain,   such    as 
Ammon's  horns,  cerebellum,  cerebrum  and  medulla  are  fixed  in 
equal  parts  of  formalin  and  95%  alcohol  for  12  to  18  hours.     They 
are  then  treated  as  follows: 

1.  95%  alcohol,  y2  hour. 

2.  95%  alcohol  again  for  %  hour. 

3.  Anilin  oil  until  clear ;  one  hour  is  usually  sufficient. 

4.  Xylol  15  to  30  minutes. 

5.  Melted  paraffin,  2%  hours. 

6.  Cut  to  3  or  4  microns. 

7.  Stain  in  hematoxylin  and  eosin. 

Cell  inclusions  known  as  negri  bodies,  which  are  usually  found 
most  abundant  in  Ammon's  horns,  are  the  evidence  of  rabies. 

The  medulla  is  searched  for  infiltrations  around  the  blood  ves- 
sels. These  changes  are  found  in  a  few  conditions  other  than 
rabies. 

B.  Smear  Method.1 

a.  Small  pieces  of  the  various  parts  of  the  brain  are  crushed  out 
between  two  clean  slides  and  treated  as  follows: 

1.  Fix  the  smears  while  still  wet  in  methyl  alcohol  (neutral- 

ized with  sodium  carbonate)   to  which  yV  %  °^  picric 
acid  has  been  added.     Blot  off  excess  of  fixative. 

2.  Stain  smears  as  follows: 

Saturated  alcoholic  solution  of  fuchsin,  3  cc. 
Saturated  alcoholic  solution  of  methylene  blue,  2  cc. 
Distilled  water,  30  cc. 

Heat  the  slide  over  a  flame  until  it  steams,  wash  in  tap 
water  and  blot.     The  stain  does  not  keep  long. 

1  Williams,  Amer.   Jour,   of  Public  Hygiene,  Feb.,   1908. 


356  MEDICAL  BACTERIOLOGY 

The  negri  bodies  are  often  found  outside  of  the  cell  and  can  be 
recognized  by  their  form,  color,  and  internal  structure. 

C.     Pasteur 's  Method. 

a.  The  medulla  of  the  suspected  animal  is  removed  under  aseptic 
precautions,  as  soon  as  possible  after  death. 

b.  Place  a  piece  of  the  medulla  about  the  size  of  a  pea,  in  4  or 
5  cc.  of  sterile  bouillon  and  thoroughly  grind  up  the  same. 

c.  Anesthetize  a  rabbit  with  ether,  clip  the  hair  from  between 
the  eyes  and  ears  and  disinfect  with  a  carbolic  acid  solution. 

d.  Make  a  longitudinal  incision  through  the  skin  and  subcutan- 
eous tissue  along  the  median  line,  while  a  crucial  incision  is  made 
through  the  periosteum  on  one  side  of  median  line  thus  avoiding 
hemorrhage  from  the  longitudinal  sinus.     The  periosteum  is  then 
pushed  back  and  a  disc  of  the  skull  (14  inch  in  diameter)  removed 
with  a  trephine  and  the  dura  mater  exposed. 

e.  With  a  sterile  hypodermic  syringe  introduce  2  or  3  drops  of 
the  suspension  of  medulla  beneath  the  dura  mater,  stitch  the  skin, 
dry,  and  seal  the  wound  with  collodion. 

The  rabbits  apparently  experience  no  inconvenience ;  the  wound 
heals  rapidly  and  the  rabid  symptoms  appear  in  from  15  to  30  days, 
although  sometimes  they  may  occur  earlier  or  much  later. 

EXERCISE  107.     EXAMINATION  OF  MATERIAL  FROM  HUMAN 

AUTOPSIES. 

At  human  autopsies  smears  from  the  organs  should  be  made  on 
cover-glasses  and  afterwards  stained  and  examined.  Plate-cultures 
should  also  be  made  from  the  various  organs.  In  all  cases  the  sur- 
face from  which  the  material  is  to  be  obtained  should  first  be  burned 
to  avoid  infection  of  cultures  with  extraneous  germs.  Portions  of 
the  various  organs  should  also  be  preserved  and  hardened  in  alcohol. 


358  MEDICAL  BACTERIOLOGY 


CHAPTER  X 

DETECTION  OF  PATHOGENIC  BACTERIA  IN 
WATER  AND  MILK  SUPPLIES 


EXERCISE  108.     EXAMINATION  OF  WATER  FOR  PATHOGENIC 

BACTERIA. 

It  is  rarely  necessary  to  test  water  directly  for  either  the  typhoid 
or  cholera  organisms,  as  there  is  little  chance  of  their  being  found 
except  in  the  most  grossly  polluted  waters.  What  is  usually  sought 
for  is  evidence  of  sewage  pollution.  If  this  is  found  the  water  is 
not  regarded  as  potable.  The  more  common  methods  of  detecting 
fecal  bacteria  have  already  been  given  (Chapter  V).  The  follow- 
ing methods  are  reliable  and  the  detection  of  these  germs  in  artifi- 
cially infected  waters  furnish  most  excellent  practice  for  the  student. 

Bacillus  typhosus.  In  the  examination  of  water  it  is  best  to  con- 
centrate the  bacteria  by  filtering  a  large  amount  of  the  water  through 
a  Berkefeld  filter  and  to  use  the  slime  on  the  filter  to  make  the  plates. 

a.  Parietti's  method,  see  103. 

~b.  Hiss'  method.  Make  plate  cultures  and  incubate  at  38°  C. 
for  18  hours.  Inoculate  suspicious  colonies  into  Hiss '  tube  medium, 
fermentation  tube  and  milk.  Also  make  indol  test  and  try  Widal 
reaction. 

c.  Animal  Inoculation.     (Michigan  method.) 

1)  Inoculate  suspected  water  into  bouillon  tubes  or  flasks,  and 
incubate  at  38°  C. 

2)  Twenty- four  to  forty-eight  hours  later  inoculate  one  cc.  into 
the  peritoneal  cavity  of  a  white  rat. 

3)  If  animal  recovers  B.  typhosus  is  not  present.     If  animal  dies 
hold  autopsy  and  isolate  and  study  organism  causing  death. 

Microspira  comma. 

a.  If  there  be  a  reason  to  believe  that  the  spirilla  are  very  numer- 
ous gelatin  plate  cultures  can  be  made  directly  from  the  water,  and 
the  suspicious  colonies  worked  up. 


360  MEDICAL  BACTERIOLOGY 

1}.  Ordinarily  the  organisms  are  very  sparse  and  large  quantities 
must  be  used.  100-1000  cc.  are  placed  in  flasks  and  \%  of  peptone 
and  0.5%  salt  are  added,  the  fluid  made  alkaline  and  incubated  at 
38°  C.  for  6-24  hours.  Then  gelatin  plate  cultures  are  made  from 
the  upper  layers  and  the  suspicious  colonies  worked  up  as  above. 

Here  and  in  typhoid  the  agglutination  of  the  germ  with  great 
dilution  of  a  high  potency  serum  is  the  crucial  test. 

Bacterium  anthracis  (Robert's  Method.) 

a.  Heat  suspected  water  to  80°  for  ten  minutes  to  kill  water 
bacteria. 

Z>.  Make  plates  in  agar  and  in  gelatin  and  work  up  colonies. 

c.  Inoculate  a  guinea  pig  with  several  cubic  centimeters  of  the 
water. 

REFERENCES.     Horrocks  and  Prescott  &  Winslow. 

EXEECISE  109.     EXAMINATION  OF  MILK  FOB  PATHOGENIC 

BACTEEIA. 

Bacterium  diphtheriae. 

Where  Bacterium  diphtheriae  is  suspected  in  milk,  make  a  con- 
siderable number  of  streak  cultures  on  Loeffler's  blood  serum  and 
incubate  at  38°  C.  from  8  to  12  hours,  stain  and  examine  micro- 
scopically. 

Bacterium  tuberculosis. 

Hammond's  method  of  examining  milk  for  B.  tuberculosis.  See 
Sputum,  101. 

Animal  Inoculation. 


USE  OF  MAIL  FOR  TRANSMISSION  OF  BACTERIA. 

Concerning  the  transmission  of  material  containing  bacteria  in  mails,  see 
Postal  Guide,  1898,  Euling  No.  82,  p.  901,  part  of  which  is  as  follows:  "That 
the  order  of  the  Postmaster-General  of  June,  1893,  forbidding  the  use  of 
mails  for  the  transmission  of  specimens  of  germs  of  cholera  or  other  diseased 
tissues,  is  hereby  modified  to  this  extent:  Specimens  of  diseased  tissue  may 
be  admitted  to  the  mails  for  transmission  to  United  States,  State  or  munic- 
ipal laboratories  only  when  inclosed  in  mailing  packages  constructed  in 
accordance  with  the  specifications  hereinafter  enumerated.  Upon  the  outside 
of  every  package  shall  be  written  or  printed  the  words:  ' Specimen  for  Bac- 
teriological examination/  No  package  containing  diseased  tissue  shall  be 
delivered  to  any  representative  until  a  permit  shall  have  first  been  issued  by 
the  Postmaster-General,  certifying  that  said  institution  has  been  found  to 
be  entitled,  in  accordance  with  the  requirements  of  this  regulation,  to  receive 
such  specimens." 


APPENDIX  A 

A  KEY  TO  THE  IDENTIFICATION  OF  THE  COMMON  PATHOGENIC 
AND  A  FEW  OF  THE  WELL  KNOWN  SAPEOPHYTIC  BACTERIA 


This  key  has  been  compiled  from  the  works  of  Migula  and  Chester,  the 
latter  of  which  contains  a  very  complete  key  to  practically  all  known  bac- 
teria. To  this,  as  well  as  to  Migula,  Sternberg  and  Kolle  and  Wassermann 
(for  the  pathogenic  bacteria),  the  student  is  referred  for  detailed  descriptions 
of  the  various  organisms. 

Cells  in  their  free  condition  globular  (cocci). 
A.  Cells  without  flagella. 

I.  Division  in  only  one  direction  of  space  forming  chains  (streptococci). 

1.  Grow  on  gelatin. 

a)   Do  not  liquefy  gelatin. 

1)   No  surface  growth  in  gelatin  stabs. 

Sir.  erysipelatos  Fehleisen. 

II.  Division  in  two  directions  of  space  (micrococci). 

1.  Grow  on  gelatin. 

a)  Colonies  white. 

1)  Do  not  liquefy  gelatin. 

M.  Melitinsis  Bruce. 

2)  Liquefy  gelatin. 

M.  pyogenes  var.  albus  ( Eosenbach )  L.  &  N. 

b)  Colonies  yellow,  and  liquefy  gelatin. 

M.  pyogenes  var.  awews(Rosenbach)L.  &  N, 

2.  Do  not  grow  on  gelatin. 

M .  gonorrhoeae  (Baum)  Fluegge. 
M.  Weichselbaumii  (Trevisan). 

III.  Division  in  three  directions  of  space  (sarcinae). 
1.  Grow  on  gelatin. 

a)  Colonies  white. 

1)   Do  not  liquefy  gelatin. 

Sar.  tetragena  (Gaffky)  Mig. 

b)  Colonies  yellow. 

1)  Do  not  liquefy  gelatin. 

Sar.  lutea  Fluegge. 
Sar.  ventriculi  Goodsir. 

2)  Liquefy  gelatin. 

Sar.  aurantiaca  Fluegge. 

Cells  short  or  long,  cylindrical,  straight,  without  sheath,  endospores  present 
or  absent,  non-motile  (bacteria). 

361 


362  APPENDIX  A. 

A.  Forms  endospores. 

I.  Grow  at  room  temperature. 
1.  Gelatin  liquefied. 

Bact.  anthracis  (Koch)  Mig. 

B.  Without  endospores. 

I.  Aerobic  and  facultative  anaerobic. 

1.  Grow  well  on  gelatin  and  do  not  liquefy  it. 

a)  Gram's  stain  negative. 

1)  Gas  generated  in  dextrose  media. 

i)   Gas  generated  in  lactose  media. 

Bact.  aerogenes  (Esch.)  Mig. 

Bact.  capsulatum  (Sternberg)  Chester, 
ii)  Little  or  no  gas  in  lactose  media. 

Bact.  pneumonicum  (Fried.)  Mig. 

2)  No  gas  in  dextrose  media. 

Bact.  cholerae  (Zopf)  Kitt. 
Bact.  bovisepticum  (Kruse)  Mig. 
(see  also  B.  pestis) 

b)  Gram's  stain  positive. 

1)  Gas  generated  in  dextrose  media. 

Bact.  acidi-lactici  Hueppe. 
Bact.  phosphor  esc  ens  (Cohn)  Fischer. 

2)  No  gas  in  dextrose  media. 

Bact.  rhinoscleromatis  (Trevisan)  Mig. 

2.  Gelatin  liquefied  slowly. 

Bact.  mallei  (Loeffler)  Mig. 
Bact.  rhusiopathiae  (Kitt)  Mig. 

3.  Do  not  grow  well  on  gelatin  at  room  temperature. 

a)  Stain  with  basic  aniline  dyes  but  are  readily  decolorized  by 

mineral  acids  when  stained  with  carbol-fuchsin. 

1)  Grow  well  in  bouillon  at  body  temperature  and  stain  by 

Gram's  method. 

Bact.  diphtheriae  (Loeffler)  Mig. 

Bact.  pseudodiphtheriticum  (Loeffler)  Mig. 

2)  Do  not  grow  in  bouillon  or  on  ordinary  media. 

Bact.  leprae  (Hansen)  Mig. 

3)  Growth  very  limited  on  ordinary  media. 

i)   Gram's  stain  positive. 

Bact.  pneumoniae  (Weichsel.)  Mig. 
ii)   Gram's  stain  negative. 

Bact.  inftuenzae  (Pfeiffer)  L.  &  N. 

b)  Do  not  stain  with  aqueous  solutions  of  basic  aniline  dyes  and 

not  easily  decolorized  by  acids. 

Bact.  tuberculosis  (Koch)  Mig. 
Bact.  tuberculosis  var  avium  (Kruse)  Mig. 
II.  Obligate  anaerobic. 

Bact.  Welchii  Mig. 

Cells  short  or  long,  cylindrical,  straight,  without  sheath,  endospores  present 
or  absent,  motile,  nagella  distributed  over  whole  body  (bacilli). 


APPENDIX  A  363 

A.  Form  endospores. 

I.  Aerobic  or  facultative  anaerobic. 

1.  Potato  cultures  irregularly  wrinkled. 

B.  vulgatus  Trevisan. 
4.  Potato  cultures  smooth. 

B.  sub  tills  (Ehrenb.)  Cohn. 
I.  Obligate  anaerobes. 

1.  Eods  not  swollen  at  sporulation. 

B.  oedematis  Zopf. 

2.  Eods  spindle-shaped  at  sporulation. 

B.  Feseri  (Trevisan)  Chester. 
B.  botulinus  v.  Ermengen. 

3.  Eods  clavate-capitae  at  sporulation. 

B.  tetani  Nicolaier. 

B.  Spore  formation  not  observed. 

I.  Aerobic  or  facultative  anaerobic. 

1.  Gelatin  colonies  roundish  not  distinctly  ameboid. 

a)  Gelatin  not  liquefied. 

1)   Gram's  stain  negative. 

i)  Generate  gas  in  dextrose  media. 
*  Coagulate  milk. 

§  Produce  indol. 

B.  coll  (Escherich)  Mig. 
§§  Do  not  produce  indol. 

B.  enteritidis  Gaertner. 
**  Do  not  coagulate  milk. 

B.  Salmonii  (Trevisan)  Chester. 
B.  icteroides  Sanerelli. 

ii)   Gas  not  generated  in  dextrose  media. 
B.  typhosus  Zopf. 
B.  dysenteriae  Shiga. 
B.  peslis  L.  &  N. 

b)  Liquefy  gelatin. 

1)  Generate  gas  in  dextrose  media. 

B.  cloacae  Jordan. 

2)  No  gas  generated  in  dextrose  media,  chromogenic,  pig' 

ment  reddish. 

B.  prodigiosus  (Ehrenb.)  Fluegge. 

2.  Gelatin  colonies  ameboid  or  irregular. 

a)  Do  not  liquefy  gelatin. 

B.  Zopfti  (Kurth)  Mig. 

b)  Liquefy  gelatin. 

B.  vulgaris  (Hauser)  Mig. 

Cells  cylindrical,  straight,  without  sheath,   endospores  known  in  only  few 
species.     Actively  motile,  flagella  attached  to  the  poles  (pseudomonas) . 

A.  Produce  a  greenish-bluish  fluorescence  in  the  culture  media. 
I.  Gelatin  liquefied. 


364  APPENDIX  A 

1.  Milk  coagulated. 

Ps.  aeruginosa  (Schroeter)  Mig. 

2.  Milk  not  coagulated. 

P$.  ftuorescens  (Fluegge)  Mig. 
II.  Gelatin  not  liquefied. 

1.  Milk  rendered  alkaline. 

Ps.  syncyanea  (Ehrenb.)  Mig. 

2.  Milk  reaction  not  changed. 

Ps.  putrida  (Fluegge)  Mig. 

Cells  cylindrical,  more  or  less  spirally  curved,  without  endospores;  actively 

motile,  flagella  attached  to  the  poles  (microspira) . 
A.  Liquefy  gelatin. 

I.  Produce  indol  in  24  hours. 

1.  Very  pathogenic  to  pigeons.. 

Microspira  Metschnikovi  (Gamaleia)  Mig. 
Microspira  $c7mt//A;t7/tewsis(  Abbott)  Chester. 

2.  Not  distinctly  pathogenic  to  pigeons. 

Microspira  comma  (Koch)  Schroeter. 

II.  Little  or  no  indol  in  24  hours. 

Microspira  Finklerii  Schroeter 

Cells  in  their  ordinary  form  long  branched  filaments;  cultures  generally  have 
a  mouldy  appearance. 

A.  Gelatin  liquefied. 

Streptothrix  bovis  (Harz)  Chester. 

B.  Gelatin  not  liquefied. 

I.  No  distinct  pigment  on  gelatin  or  agar. 

Streptothrix  farcinica  Rossi-Doria. 

II.  Growths  on  gelatin  or  agar  become  reddish. 

Streptothrix  madurae  Vincent. 
Threads  without  distinct  sheaths. 

A.  Without  sulphur  grains. 

Lepothrix  buccalis  Miller. 

B.  With  sulphur  granules,  motile,  not  attached. 

Beggiato  alba  (Vaucher)  Trevisan. 

Threads  with  sheaths. 

A.  Without  sulphur  granules. 

I.  Without  pseudodichotomous  branching. 

Crenothrix  polyspora  Cohn. 

II.  With  pseudodichotomous  branching. 

1.  Growths  on  gelatin  whitish  but  gelatin  stained  brown. 

Cladothrix  dichotoma  Cohn. 

2.  Gelatin  not  stained  brown,  colonies  floccose— filamentous. 

Cladothrix  intrica  Eussell. 

B.  With  sulphur  granules. 

Thiothrix  tenuissima  Winogradsky. 


APPENDIX  B 


Name  of  organism,  source,  habitat,  etc. 


REFERENCES. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


6.  Agrar. 


c.  Gelatin. 


d.  Other  media. 


2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 
&.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT Y  : 

a.  Character  of  movement.. 
&.  Flagella  stain 


5.    SPORES: 


SPECIAL.  CHARACTERS: 

a.  Capsules 

&.  Involution  forms 

c.  Deposits  or  vacuoles.. 

d.  Pleomorphism 


365 


CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  _j-   or  - 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48hoursat °C.        I       6  days  at *C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at CC. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


APPENDIX  B 


367 


Gelatin  Stab:    Grown  24  hours  at °C. 


O 


48  hours  at 


6  days  at ,C\ 


Agar  Streak:     Grown  24  hours  at 


48  hours  at . . 


6  days  at °C. 


Potato:    Grown  24  hours  at °C.  Q| 

n 
h 

48  hours  at °C.  6  days  at °C. 

i 

Bouillon:     Grown  24  hours  at °C. 

2 

\ 
48  hours  at »C.  6  day  sat... 


368  PHYSIOLOGICAL  CHARACTERS 


1,  RELATION  TO  TEMPERATURE: , 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION:. 


5.  GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia. . , 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODPR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


APPENDIX  B 


369 


Name  of  organism,  source,  habitat,  etc. 


REFEBENCES. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


i.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


b.  Agar. 


e.  Gelatin. 


d.  Other  media. 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian-violet. . 

b.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILIT  Y  : 

a.  Character  of  movement.. 

b.  Flagella  stain 


5.    SPORES: 


SPECIAL  CHARACTERS: 

a.  Capsules 

b.  Involution  forms 

c-.  Deposits  or  vacuoles.. 
d.  Pleomorphism , 


370  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  4. 


or 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at "C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at °C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


APPENDIX  B 


371 


Gelatin  Stab:     Grown  24  hours  at. 


48  hours  at 


(lays  at 


Agar  Streah:     Grown  24  hours  at 


48  hours  at 


days  at "C. 


Potato :    Grown  24  hours  at °C. 


\ 


48  hours  at 


6  days  at.. 


Bouillon:     Grown  24  hours  at. 


48  hours  at °C. 


6  days  at.. 


372  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN  : 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc. :— . 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm- 

gas  formula,  H:  COz:  : : 

&.  lactose c.  saccharose 

6.  ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic. . .  


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : . , 


APPENDIX  B 


373 


Name  of  organism,  source,  habitat,  etc. 


REFERENCES. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


ft.  Agar. 


c.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian-violet. . 
ft.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY: 

a.  Character  of  movement., 
ft.  Flagella  stain 


5.    SPORES: 


6.    SPECIAL  CHARACTERS: 

a.  Capsules 

ft.  Involution  forms 

c.  Deposits  or  vacuoles.. 

d.  Pleomorphism 


374  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -j_   


Gelatin  plate:    Grown  24  hours  at °C. 

[a)  Surface  Colonies  (b)  Deep  Colonies, 


Sketches, 


48  hours  at , ..........  °C, 

; 


6  days  at.,.,...,,...,,. *C. 


Agar  plate;    Grown  24  hours  at. 


(a)  Surface  Colonies, 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


APPENDIX  B 


375 


Gelatin  Stab:     Grown  24  hours  at °C. 


Agar  Streak:     Grown  24  hours  at , 


48  hours  at                     °C 

I 


a!/ 


48  hours  at . . 


6  days  at °C. 


Potato:     Grown  24  hours  at 


48  hours  at 


6  days  at. 


Bouillon:     Grown  24  hours  at °C. 


48  hours  at "C. 


6daysat "C. 


376  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: « t 

optimum °C. ;  limits to °C. ; 

thermal  death-point °C. ;  time  of  exposure minutes 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION; 


6.      GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture : 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent..  48  hours percent. 

72  hours per  cent .hours per  cent, 

reaction  in  open  arm 

gas  formula,  H:  COa:  : : 

6.  lactose c.  saccharose 

6.    ACID  OR  ALKALI  PRODUCTION:...  . 


litmus  milk 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) ; 


APPENDIX  B 


377 


Name  of  organism,  source,  habitat,  etc. 


REFERENCES. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


&.  Agrar- 


c.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet. . 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 


5.    SPORES: 


6.    SPECIAL  CHARACTERS:.... 

a.  Capsules 

6.  Involution  f orms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism . 


378  CULTURE  CHARACTERS 

Eeaction  of  media  (Fuller's  scale)  -f_ or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48hoursat  .........................  °C.  6  days  at 


«C. 


Agar  plate;    Grown  24  hours  at °C.       I     Sketches. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


48  hours  at °C. 


6  days  at *C. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum/ 


APPENDIX  B 


379 


Gelatin  Stab:     Grown  24  hours  at  °C                  ^j  < 

. 

9 

i 
\       / 

hours  at  °C 

u 

c 

*->  •       \ 

OS    ,/ 

2 

!^ 

Q 

O 

Z 

2 
3 

o 

A 

A 

U 

48  hours  at                                     °C                                     6  days  at        

0 

c. 

Potato  :     Grown  24  hours  at  °C 

;A 

.a 

hours  at  °( 

/\ 

/    \ 
/       \ 

w 

o 

c 

Bouillon:     Grown  24  hours  at     °C                 r\ 

. 

* 

X 

A 

O 

I 

V  J 

48  hours  at  °C.                                  6  days  at.  .  . 

0 

c. 

380  PHYSIOLOGICAL  CHARACTERS 


1.  RELATION  TO  TEMPERATURE: 

optimum °C. ;  limits to °C.; 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN:... 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent.,  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  CCb:  : : 

6.  lactose c.  saccharose 

6.  ACID  OR  ALKALI  PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OP  NITRATES: 

to  nitrites to  ammonia.., 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: .  . 


proteolytic 

digestion  of  gelatin digestion  of  casein. 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


APPENDIX  B 


381 


Name  of  organism,  source,  habitat,  etc. 


REFERENCES. 


MORPHOLOGICAL  CHARACTERS  : 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


&.  Agrar. 


c.  Gelatin. 


d.  Other  media., 


2.  SIZE: 

3.  STAINING  POWERS  : 

a.  Aqueous  gentian-violet.. 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILJTY  : 

a.  Character  of  movement.. 
&.  Flagella  stain 


5.    SPORES: 


6.    SPECIAL  CHARACTERS: 

a.  Capsules 

6.  Involution  forms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism 


382  CULTURE  CHARACTERS 

Eeactiou  i-  media  (Fuller's  scale)  -{- or  — 


Gelatin  plate:    Grown  24  hours  at °C. 


(a)  Surface  Colonies. 


(b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at »C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surf  ace  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  Hours  at °C. 


6  days  at 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


APPENDIX  B 


383 


Gelatin  Stab:     Grown  24  hours  at CC. 


48  hours  at 


6  days  at <>C. 


Streak:     Grown  24  hours  at °C. 


O 


48  hours  at 


6  days  at °C. 


Potato :     Grown  24  hours  at °C. 


Bouillon:     Grown  24  hours  at. 


•C. 


48  hours  at                                   °C 

6  days  at                           °C 

48  hours  at °C. 


6  days  at. 


384  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: , , 

optimum °C. ;  limits to °£. ; 

thermal  death-point °C.;  time  of  exposure minutes; 

medium  in  which  exposure  is  made 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


6.      GAS  PRODUCTION  IN   SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2)  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours per  cent.,  48  hours percent. 

72  hours per  cent., hours per  cent. 

reaction  in  open  arm 

gas  formula,  H:  COz:  : : 

6.  lactose c.  saccharose 

6.     ACID  OR  ALKALI   PRODUCTION: 


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia.. . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) :. 


APPENDIX  B 


385 


Name  of  organism,  source,  habitat,  etc. 


REFERENCES. 


MORPHOLOGICAL  CHARACTERS: 


SKETCHES. 


1.    FORM  AND  ARRANGEMENT: 
a.  Bouillon 


b.  Agar. 


c.  Gelatin. 


d.  Other  media.. 


2.  SIZE: 

3.  STAINING  POWERS: 

a.  Aqueous  gentian- violet.. 
6.  Loeffler's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

4.  MOTILITY  : 

a.  Character  of  movement.. 
6.  Flagella  stain 


.    SPORES:. 


6.    SPECIAL  CHARACTERS:.... 

a.  Capsules '. 

6.  Involution  forms 

c.  Deposits  or  vacuoles. 

d.  Pleomorphism 


386  CULTURE  CHARACTERS 

Keaction  of  media  (Fuller's  scale)  _- 


or  — 


Gelatin  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at «C. 


Agar  plate:    Grown  24  hours  at °C. 

(a)  Surface  Colonies.  :   (b)  Deep  Colonies. 


Sketches. 


48  hours  at °C. 


6  days  at. 


Special  Media:    (Such  as  litmus  milk  and  blood  serum.) 


APPENDIX  B 


387 


Gelatin  Stab:    Grown  24  hours  at °C. 


48  hours  at °C. 


6  days  at 


Agar  Streak:     Grown  24  hours  at °C. 

o  o 

/\ 

/ 

I     ; 

48  hours  at °C.  6  days  at CC. 

Potato:     Grown  24  hours  at °C.  >j 

/      \ 

«  /  \       a 

i  i  I 

y 

48  hours  at °C.  6  days  at °C. 

Bouillon:     Grown  24  hours  at. 


48  hours  at °C. 


6  days  at °C. 


388  PHYSIOLOGICAL  CHARACTERS 

1.  RELATION  TO  TEMPERATURE: 

optimum ° C. ;  limits to °C.: 

thermal  death-point °C. ;  time  of  exposure minutes; 

medium  in  which  exposure  is  made » 

2.  RELATION  TO  FREE  OXYGEN: 


3.    RELATION  TO  OTHER  AGENTS,  SUCH  AS 

desiccation,  light,  disinfectants,  etc.:—. 


4.    PIGMENT  PRODUCTION: 


5.  GAS  PRODUCTION  IN  SUGAR  MEDIA: 

a.  dextrose  (1)  Shake  culture: 

(2*  Fermentation  tube,  growth  in  open  arm closed  arm 

rate  of  development:  24  hours percent..  48  hours percent. 

72  hours per  cent hours per  cent. 

reaction  in  open  arm 

gas  formula,  H :  COs :  : : 

v.  lactose c.  saccharose • 

6.  ACID  OR  ALKALI   PRODUCTION:...  


litmus  milk. 


7.  REDUCTION  OF  NITRATES: 

to  nitrites to  ammonia. . . 

8.  INDOL  PRODUCTION 

48  hours days . 

9.  ENZYME  PRODUCTION: 


proteolytic 

digestion  of  gelatin digestion  of  casein . 

diastatic 


10.  CHARACTERISTIC  ODOR: 

11.  PATHOGENESIS  (or  other  special  characters) : 


APPENDIX  C 


TABLES 
COMPARISON  OF  THERMOMETERS. 

(From  GQiild's  Dictionary  of  Medicine) 


a 
I 

I 

ID 
•< 
H 
« 

A 

1 

H 
0 

p 

« 
I 

H 

o 

p 

212 

100 

80 

122 

50 

40 

32 

0 

0 

210 

98.9 

79.1 

120 

48.9 

39.1 

30 

-1.1 

-0.9 

208 

97.8 

78.2 

118 

47.8 

38.2 

28 

-2.2 

-1.8 

206 

96.7 

77.3 

116 

46.7 

37.3 

26 

-3.3 

-27 

204 

95.6 

76.4 

114 

45.6 

36  4 

24 

-4.4 

-3.6 

202 

94.4 

75.6 

112 

44.4 

35.6 

22 

-5.6 

-4.4 

200 

93.3 

74.7 

110 

43.3 

34.7 

20 

-6  7 

-5.3 

198 

92.2 

73.8 

108 

42.2 

33.8 

18 

-7.8 

-6.2 

196 

91.1 

72.9 

106 

41.1 

32.9 

16 

-8  9 

-7.1 

194 

90 

72 

104 

40 

32 

14 

-10 

-8 

192 

88.9 

71.1 

102 

38.9 

31.1 

12 

-11  1 

-8.9 

190 

87.8 

70.2 

100 

37  8 

30.2 

10 

-12.2 

-9.8 

188 

86.7 

69.3 

98 

36.7 

29.3 

8 

-13.3 

-10.7 

186 

85.6 

68.4 

96 

35.6 

28.4 

6 

-14.4 

-11.6 

184 

84.4 

67.6 

94 

34.4 

27.6 

4 

-15.6 

-12.4 

182 

83.3 

66.7 

92 

33.3 

26.7 

2 

-16.7 

-13.3 

180 

82.2 

65.8 

90 

32.2 

25.8 

0 

-17.8 

-14.2 

178 

81.1 

64.9 

88 

31.1 

24.9 

-2 

-18.9 

-15.1 

176 

80 

64 

86 

30 

24 

-4 

-20 

-16 

174 

78.9 

63.1 

84 

28.9 

23.1 

-6 

-21.1 

-16.9 

172 

77.8 

62.2 

82 

27.8 

22.2 

-8 

-22.2 

-17.8 

170 

76.7 

61.3 

80 

26.7 

21.3 

-10 

-23.3 

-18.7 

168 

75.6 

60.4 

78 

25.6 

20.4 

-12 

-24.4 

-19.6 

166 

74.4 

59.5 

76 

24.4 

19.6 

-14 

-25  6 

-20.4 

164 

73.3 

58.7 

74 

23.3 

18.7 

-16 

-26.7 

-21.3 

162 

72.2 

57  8 

72 

22.2 

17.8 

-18 

-27.8 

-22.2 

160 

71.1 

56.9 

70 

21.1 

16.9 

-20 

-28.9 

-23.1 

158 

70 

56 

68 

20 

15 

-22 

-30 

-24 

156 

68.9 

55.1 

66 

18.9 

15.1 

-24 

-31  1 

-24.9 

154 

67.8 

54.1 

64 

17.8 

14.2 

-26 

-32.2 

-25.8 

152 

66.7 

53.3 

62 

16.7 

13.3 

-28 

—33  .  3 

-26.7 

150 

65.6 

52.4 

60 

15.6 

12.4 

-30 

-34.4 

-27.6 

148 

64.4 

51.6 

58 

14.4 

11.6 

-32 

-35.6 

-28.4 

146 

63.3 

50.7 

56 

13.3 

10.7 

-34 

-36.7 

-29.3 

144 

-  62.2 

49.8 

54 

12.2 

9.8 

-36 

-37.8 

-30.2 

142 

61.1 

48.9 

52 

11.1 

8.9 

-38 

-38.9 

-31.1 

140 

60 

48 

50 

10 

8 

-40 

-40 

-32 

138 

58.9 

47.1 

48 

8.9 

7.1 

-42 

-41.1 

-32.9 

136 

57.8 

46.2 

46 

7.8 

6.2 

-44 

-42.2 

-33.8 

134 

56.7 

45.3 

44 

6.7 

5.3 

-46 

-43.3 

-34.7 

132 

55.6 

44.4 

42 

5.6 

4.4 

-48 

-44.4 

-35.6 

180 

54.4 

43.6 

40 

4.4 

3.6 

-50 

-45.6 

-36.4 

128 

53.3 

42.7 

38 

3.3 

2.7 

-52 

-46.7 

-37.3 

126 

52.2 

41.8 

36 

2.2 

1.8 

-54 

-47.8 

-38.2 

124 

51.1 

40.9 

34 

1.1 

0.9 

-56 

-48.9 

-39.1 

To  change  Centigrade  to  Fahrenheit:  (C  X  f )  +  32  =  F. 

For  example,  to  find  the  equivalent  of  10°  Centigrade,  C  =  10°  (10°  X  f) 
+  32  =  50°  F. 

To  change  Fahrenheit  to  Centigrade:  (F.  —  32°)  X  f  =  C. 

For  example,  to  reduce  50°  F.  to  Centigrade,  F.  =  50°  and  (50°  —  32°) 
X  f  =  10°  C.  or  —  40°  F.  to  Centigrade,  F.  =  —  40°  (—  40°  —  32°)  =  —  72°, 
whence  —  72°  X  f  =  —  40°  C. 

389 


390  APPENDIX  C 

COMPARATIVE  LIST  OF  METRIC  AND  ENGLISH  SYSTEMS. 

Metre  =  100  centimetres,  1000  millimetres,  =  39.3704  inches. 

Millimetre  =  1000  microns,  -£%  inch,  approximately. 

Inch  =  25.399772  mm.   (25.4  approximately). 

Litre  =  1000  millilitres  or  1000  cc.,  1  quart  (approximately). 

Cubic  Centimetre  =  y^V^  of  a  litre. 

Fluid  ounce  (8  fluid  drachms)  =  29.578  cc.,  (30  cc.,  approximately). 

Gram  =  15.432  grains. 

Kilogram  =  2.204  avoirdupois  pounds  (2^  pounds,  approximately). 

Ounce,  avoirdupois,  =(437^  grains) =28. 349  grams  )  30  grams,   ap* 

Ounce,  Troy  or  apothecaries,  =(480  grains)  =31. 103  grams,  )  proximately. 


INDEX 


ABBE,  condenser,  34. 
Absorbent  cotton,  14. 
Acetic  acid,  decolorizing  action,  60. 
Acid 

acetic,  60. 

alcohol,  312,  316. 

carbolic,  use  in  detecting  enzymes,  80. 
in  Pariette's  solution,  338. 

production,    76. 

pyrogallic,   272. 

sulphanilic,   76. 
Acids,  determination  of,  76. 
Acidity  of  Media,  8. 
Actinomyces  bovis,  263. 
Aerobes,  72,  74. 

pathogenic,    138. 
Agar  culture  medium,  16. 

dextrose,   64. 

filtering  of,  16. 

hanging-drop  culture,  44. 

lactose,  64. 

melting  point  of,  56. 

plate  cultures,  56. 

preparation  of,  16. 

slopes,  16. 

sterilization  of,  16. 
Air  analysis.  126. 

pump,  14. 

Alcohol,  as  a  decolorizing  agent,  60. 
Alcoholic   solutions  of  dyes.   28. 
Alkalies,  detection  of  in  cultures,.  76. 
Alkalinity  of  media,  8. 
Ameba  coli.  344.  352. 
Ammonia  in  cultures,  78. 
Anaerobes,   72,   74. 

pathogenic,  92,  271. 

Wright's  Method  of  Cultivating,  272. 
Analysis  of  Air,  126. 

of  gas,   74. 

of  milk,  132. 

of  soil,  132. 

of  wa.er,  128, 
Anilin  dyes,  28. 

water,  30. 
Animal  autopsy,  307. 

cage,  300. 

care  of  inoculated,  300. 

experiment,   blank  for,   308. 

inoculation,  294. 
Anthrax  bacillus,  1^7. 

group,  91,   167. 
Antiseptic  action,  72. 
Antiseptics,  testing  of,  134. 
Apparatus  for  cooling  plates,  54. 

for  tubing  media,  10. 
Arnold  sterilizer.  10. 
Arrangement  of  bacteria,  91. 
Asiatic  cholera  germ,  251. 
Asparagin,  46. 
Aspergillus,   50. 
Aspirator,  126. 
Autoclave,  12. 
Autopsy,  animal,   302. 

cultures  at,  304. 

human,  356. 

instruments,  300. 

T5ACILLTJS,  89. 
-15  acidi  lactici,  109. 

aerogenes,  175. 

capsulatus,  273. 

amylobacter,  46. 


Bacillus 

anthracis,    167. 

symptomatici,  277- 

botulinus,  285. 
bovisepticus,   187. 
capsulatum,  179. 
carbonis,  277. 
cnolerae  gallinarum,  183. 

suum,  227. 
coli,  biology  of,  219. 

effect  of  chemicals  on,   134. 
desiccation.  70. 
reaction  of  medium,   6& 

for  removing  sugars,  64. 

Gram's  stain  for,  62. 

in  water,  130. 

motility,  40,  50. 

plate  cultures,  56,  58. 

production  of  acids,  76. 
gases,  74. 

hydrogen    sulphide,    78. 
indol,   78. 

thermal  death  point,  68. 
diphtheria?,  199. 
dysenteriae,  239,  344. 
edematis,  281,  314,  318,  352. 

maiigni    381. 
enteritidis,  223. 
Feseri.  277,  314,  318. 
icteroides,   231. 
influenzas,  211. 
mallei,  191. 

mesentericus  vulgatus,  95. 
mycoides,  40,  42. 
of  blue-green  pus,  247. 
of  bubonic  plague,  243. 
of  chicken  cholera,  183. 
of  glanders    191. 
of  hemorrhagic   septicemia,   187. 
of  Japanese  dysentery,  239. 
of  malignant  edema,   281. 
of  swine  plague.  182. 
of  swine  erysipelas,  195. 
of  symptomatic  anthrax,   277. 
pestis,  243,  306.  330,  334,  352. 

bubonicae,  243. 
pneumonicae,  171. 
prodigiosus,   74,  80,  103,   108. 
proteus,  113. 
pyocyaneus,  247. 
Salmonii,  227,  306,  318,  334. 
septicemiae  hemorrhagicse,  183. 
subtilis,  24.  25,  40,  46,  48.  50,  54,  58, 

62,  66,  70,  72,  76,  80,  99. 
suipestifer,  227. 
typhi  abdominalis,  235. 
typhosus.  50,  70,  235,  318,  336,  338, 

346.  3.18. 

tetani.  46.  289,  306,  314,  352. 
tuberculosis,  see  Bact.  tuberculosis. 
vulgaris,  113. 
vulgatus.  95. 
Zopfii,  117. 
Bacteria 

arrangement  in  groups,  91. 

capsule  stain  for,  50,  314. 

cell  grouning  of,  42. 

classification,  89. 

colonies  of,   52. 

compared  with  yeasts  and  moulds,  50. 

decolorizing,  60. 

determining  size,  38. 


391 


392 


INDEX 


Bacteria 

drawing,  36. 

effect  of  chemicals  on,  72. 
of  desiccation  on,  70. 
of  direct  sunlight  on,  72. 
of  moist  and  dry  heat  on,  70. 
flagella  stain,   48. 
formation  of  ammonia  by,  78. 
of  enzymes  by.  80. 
of  gas  by,  74. 
of  indol  by,  78. 

of  sulphuretted  hydrogen  by,  78. 
involution  forms,  46. 
metachromatic   granules,   50. 
movement  of,  40. 
non-pathogenic,  93. 
pathogenic,  138,  271. 
reduction  of  nitrates  by,  76. 
relation  to  oxygen,  72. 
staining,  30. 

in  tissue,  312. 
spores   of.    46. 

thermal  death  point  of,  68. 
Bacteriacese,  89. 
Bacterium,  89. 

acidi-lactici,   109. 
jrogenes,  175. 

'anthracis,  46,  167,  304,  314,  330,  360. 
bovisepticum,  187. 
capsulatum,   50,   179. 
cholerse,  183,  316. 
cholerse  gallinarum,  183. 

suum,  227. 
coli  commune,  219. 
diphtherise,  46,  50,  199,  3C6,  314,  318, 

360. 

diagnosis,  320. 
erysipelatos  suis,  195. 
influenzse,  211. 
lactis  serogenes,  175. 
-  leprse,  314,  316,  350. 
mallei,  191,  306,  316,  332,  350. 
pestis,  243. 
phosphorescens,  121. 
pneumonise,   50,   207,    304,   314,   328, 

332,  350. 

pneumonicum,  50.  171,  304,  314. 
pseudodiphthereticum,  203,  322. 
rhusiopathise,  195,  314. 
suicida,  183. 

tuberculosis,  213,  304,  314,  316,  326, 
334,  344,  346,  350,  360. 
Gabbett's  stain,   62. 
in  sputum,  326. 
in  tissue,  316. 
in   urine,   346. 
Welchii,  273,  314,  318,  352. 
Zopfli,  117. 
Bacteriological  analysis,  126. 

diagnosis,  320. 
Beef  extract,  6. 
Beggiatoa,  90. 
Beggiatoacese,  90. 
Bismarck  brown,   28. 
Black-leg  bacillus,  277. 
Blood,  agar,  212. 

examination  of,  330. 
serum,  138. 

Boehmer's  hematoxylin,  312. 
Bolton's  potato  tubes,  18. 
Bouillon  cultures,  22,  87. 
dextrose,  64. 
lactose,  64. 
preparation  of,  6. 
saccharose,  64. 
sugar-free,  64. 
Brownian  movement,  40. 
Buccal  secretion,  320. 
Buchner's  method  of  growing  anaerobes, 

272. 
uunge's  flagella  stain,  48. 


PAGE  for  animals,  300. 

^  Calcium  chloride,  6. 

Canada  balsam,  32. 

Capsule  stain,  50,  314. 

Carbol-fuchsin,  28,  34. 

Carbolic  acid,  28,  72,  80. 

Care  of  culture  media,  18. 

Cell  grouping,  42. 

Celloidin  sections,  310. 

Centrifuge,  32#. 

Chain  coccus,  141. 

Chemicals,  antiseptic  action  of,  134. 

disinfecting  action  of,  136. 

effect  on  bacteria.  72. 
Chlamydobacteriaceae,  90. 
Cholera  group,  92,  251. 

red  reaction,  78,  344. 
Chromogenic  class,  91,  103. 
Cladothrix,  90. 
Cleaning  glassware,  2. 

slides  and  cover-glasses,  26. 

solution,    28. 
Coccaceae,  89. 

Coccus  of  Malta  fever,  153. 
Collodion  sacs,  296. 
Colon  bacillus,  see  li.  coli. 

group,  92,  219. 
Colonies,  52,  83. 

color  of,  85. 

counting,   126. 

miscroscopic  structure,  84. 

surface  elevation,  84. 

types  of,  83. 
Color  of  colonies.  85. 

production,  variation  in,  80. 
Coloring  matter,  separation  of,  108. 
Concentration  of  media,  66. 
Condensed  milk,  66. 
Condenser,  Abbe,  34. 
Cooling  apparatus,  54. 
Cornet  forceps,  32. 
Corrosive  sublimate,  IX. 
Cotton,  4. 

absorbent  for  filtering,  14. 
Counting    apparatus,    126. 
Cover-glass  preparations,   30. 
Cover-glasses,  cleaning  of,  26. 
Crenothrix,  90. 
Culture  characters  of  bacteria,  83. 

media,  6,  10,  64,  138. 

of  anaerobes,  271. 
Cultures,  agar,  22. 
plate,  52. 
roll,  56. 

bouillon,  84. 

gelatin,  stab.  22. 
plate,  52. 

hanging-drop,  44. 

incubation  of,  24. 

potato,   24 

set  of,  93. 

stab,  22. 

streak,  24. 

test-tube,  22. 

"DECOLORIZING  agents,  so. 

•*-'  Desiccation,  effect  on  bacteria,  70 
Dextrose  agar,  64. 

bouillon,  64. 

free  bouillon,   64. 

gelatin,  62. 
Diagnosis,  methods  of,  320. 

of  rabies.  354. 
Diaphragm,  iris,  36. 
Diphtheria  bacillus,  see  B.  diphtheria 

group,  92,  199. 

outfit,  320. 
Diplococcus  gonorrhoeae,  157. 

group,  91,  157. 

intracellularis  meningitldls,  159. 

pneumonise,  257. 
Disinfectants,    72,   136= 
Drawing  bacteria,  36. 


INDEX 


393 


Drigalski  &  Conradi's  medium,  342. 

Dry  air  sterilizer,  4. 

Dunham's  clearing  solution,  312. 

peptone  solution,  64. 
Dyes,  anilin,  28. 

EGG,  use  in  clearing  media,  14. 
Ehrlich's  anilin  oil  gentian  violet,  28. 
Embedding  tissue,  310. 
Emphysema  group,  92,  273. 
Endospores,  study  of,  46. 
Enzymes,  80. 
Eosin,  312. 
Ernst's  stain,  50. 
Erysipelas  group,  91,  141. 
Esmarch's  roll  cultures,  56. 
Examination  of  blood,  330. 

of  buccal  secretion,  320. 

of  feces,  338. 

of  sputum.  326. 

of  transudates  and  exudates,  346. 

of  urine,  346. 
Extract   of   beef,    6. 
Exudates,  examination  of,  346. 
Eyepieces,  see  ocular. 

FECAL  bacteria,  130. 
Feces,  examination  of,  338. 
Fermentation  tube,  74.         \ 
Filling  tubes  and  flasks,  " 
Filter,  folded,  8. 

pump,  14. 
Filtering  gelatin,  14. 

agar,  16. 

Flagella  stain,  48. 
Folder  filter,  8. 
Forceps,  Cornet,  32. 

cover-glass,   32. 

Stewart,  32. 

Form  types,  study  of,  40. 
Fraenkel's  soil  borer,  132. 
Fnedlander's  bacillus,  171. 

group,  91,  171. 
Frost's  gasometer,  74. 
Frozen  sections,  310. 
Fuchsin,  28, 

p  ABBETT'S  methylen  blue,  30. 
^   tubercle  stain,  62. 
Gas  analysis,  74. 

formula,  74. 
Gasometer,  74. 
Gelatin,  dextrose,  64. 

filtering  of,  14. 

melting  point  of,  24. 

plate  cultures,   52. 

preparation  of,  14. 

reaction  of,  14. 

sterilization  of,  14. 
Gentian  violet,  28. 
Glanders  bacillus,  191. 

group,  91,  191. 
£}lass  slides,  cleaning  of,  26. 
hollow  ground,  38. 

Jumblers,  24. 
Glassware,  cleaning  of,  2. 

sterilization  of,  4. 
Golden  pus  coccus,   149. 
Gonococcus.   157. 
Gonorrhceal  pus,  348. 
Gram's  iodine  solution,  30. 

stain,   60. 

list  of  organisms  taking  this  stain, 
314. 
Guinea  pig,  diagram,  307. 

inoculation   of,   294. 
Groups,  arangement  of  bacteria  in,  91. 

HAMMOND'S  method  of  concentrating 
tubercle  bacilli  in  milk  and  sputum, 
328. 

Hanging-drop  culture,  44. 
preparation,  38,  44. 


Hauser's  spore  stain,  48. 
Hay  bacillus,  24. 
Heat,  effect  of  on  bacteria,  68. 
Hematoxylin  stain,  312. 
Hiss'  culture  medium,  340. 
Hog  cholera  bacillus,  227. 

group,  92,  227. 
Hot  air  sterilizer,  4. 
Hydrochloric  acid,  2,  8. 
Hydrogen  generator,  271. 

TMMERSION  lens,  34. 

A    Impression  preparations,  42. 

Incubation  of  cultures,  24. 

Incubator,  24. 

Indol  test,  78,  130. 

Influenza  bacillus,  211,  328. 

group,  92,  211. 

Inoculated  animals,  care  of,  300. 
Inoculation  of  animals,  294. 

into  ear,  298. 

into  eye,  300. 

intraperitoneal,  294. 

intrapleural,   300. 

intravenous,  298. 

lymphathic,  300. 

subcutaneous,  294. 

subdural,  3o4. 

Instruments,   sterilization  of,   300. 
Involution  forms,  46. 
Iodine  solution,  30,  314. 
Iris  diaphragm,  34. 

TEFFER'S  counting  apparatus,  modified, 
J    Plate  II,  p.  137. 

TTEY  to  identification  of  bacteria,  361. 
AV  Kipp  hydrogen  generator,  271. 
Klebs-Loeffler's  bacillus,  199. 
Koch's  method  of  air  analysis,  126. 

T   ABELS,  20. 

*-*  Lactic  acid  bacillus,  109. 

Lactose  agar,  64. 

litmus  agar,   76. 
Lens  paper,  36. 
Litmus  milk,  66. 

solution,  64. 
Loeffler's  blood  serum,  138. 

methylen  blue,   28. 

stain  for  sections,  312. 
Loop,  platinum,  20. 

IVfACCONKEY'S  medium,  340. 
ltj-    Malaria,  326. 

Mallory's  stain  for  ameba  coli,  334. 
Malta  fever  group,  91,  153. 
Media,  care  of,  12,  18. 

examination  of,  12. 

labeling,  20. 

preparation,  6-18,  64,  94,  138. 

reaction  of,  8. 

sterilization  of,  10. 
Measuring  bacteria,  36. 
Mesophilic  bacteria,  68. 
Metachromatic  granules,  stain  for,  50. 
Methylen  blue,  alkaline,  28. 

Gabbett's  30. 

Loeffler's,  28. 
Mice,  white,  294. 
Micrococcus,  89. 

gonorrhceae,  157.  314,  346,  348. 

melitensis,  153. 

pyogenes,   var.   albus,   145,   314,    (see 

also  pus  micrococci). 

var.  aureus,   149,  314   (see  also  pus 

micrococci) . 

Weichselbaumii,  159,  350. 
Micrometer,  ocular,  36. 

stage,  36. 
Micron,  38. 
Microspira,   89. 

comma,  251,  342,  358, 


394 


INDEX 


Microspira 

Metschnikovi;  40,  255. 

Schulykilliensis,  259. 
Microscope,   34. 
Microscopic  study  of  plate  cultures,  58. 

of  bacteria,  40. 
Migula's  classification,  89. 
Milk  anaylsis,  132,  360. 

cultures,  87. 

litmus,  66. 

pasteurization  of,  132. 
Molecular  movement,  40. 
Monilia   Candida,   324. 
Monas  prodigiosa,  103. 
Morphology,  2,  82. 
Motility  of  bacteria,  40. 
Moulds,  morphology  of,  50. 
Mucor,  50. 

•V"EEDLE,   platinum,   20. 

f1    Neisser's  diphtheria  stain,  322. 

Nessler's  reagent,  78. 

Neutralization  of  media,  6. 

Nitrate  solution,  64. 

Nitric  acid,  decolorizing,  60. 

Nitrites,  detection  of.  76. 

Nitroso-indol   (cholera  red),  78. 

Non-pathogenic  bacteria,  93. 

Normal  solutions,  8. 

Novy's  anaerobic  jars,  271. 

QBJECTIVE,  oil-immersion,  34. 

^   Ocular  micrometer,  36. 

Ocular,   34. 

Oedema  group,  92,  277. 

Oil-immersion  ojective,  34. 

Oospora  bovis,  263. 

Oxalic  acid,  344. 

Oxygen,  relation  of  growth  to,  72. 

PARAFFIN,  sealing  tubes  with,  20. 
x      sections,  310. 
Parietti's  solution,  338. 
Pasteurization  of  milk,  132. 
Pathogenic  aerobes,  91,  138. 

anaerobes,  271. 

bacteria,   reaction  of  media  for,  8. 
best  temperature  for,   24. 
in  water  and  milk  supplies,  358. 
Penicillium,  50. 
Peptone,  6. 

Petri  dish  cultures.  52. 
Petri-Sedgwick's  air  analysis,   126, 
Pfeiffer's  capsule  bacillus,   179. 

stain,  318. 
Phenolphthalein,  8. 
Phosphorescent  class,  91,  121. 
Photobacterium  phosphorescens,  121. 
Phragmidiothrix,  90. 
Physiology  of  bacteria,  64. 
Physiological  characters  of  bacteria,  88. 
Pigeons,  inoculation  of,  294. 
Pigments,  separation  of.  107. 

variety  of,  108. 
Planococcus,  89. 
Planosarcina,  89. 
Plasmodium,  malariae,  334. 
Plasmolysis,  83. 
Plate  cultures,  agar,  56. 

gelatin,  52. 

method  of  pouring,  54. 

study  of,  58. 
Platinum   needles.   20. 
Plugging  flasks  and  tubes,  4. 
Pneumobacillus,  171. 
Pneumonia  group,  92,  207. 
Post-mortem  examination,   302. 
Potassium  hydrate.  28. 
Potassium  iodide,  30. 
Potato  bacillus,   95. 
Potatoes,  preparation  of,   18. 
Preparation  of  agar,  14. 

of  bouillon,   6. 

of  cover-glasses,  30. 


Preparation  of  gelatin,  14. 

of  hanging-drop,  38. 

of  potatoes,  18. 

of  staining  solutions,  28. 

of  water  blanks,  18. 
Proteus  vulgaris,   113. 

Zenckeri,  117. 

Pseudodiphtheria  bacillus,  203. 
Pseudomonas,   89. 

ffiruginosa,  80,  247,  314,  352. 

fluorescens,  40. 

group,  92,  247. 

violacea.  68. 

Psychrophilic  bacteria,  68. 
Piis  micrococci,  314.  324,  332,  346,  348. 
Pyrogallic  acid,  272. 

-RABBITS,  294. 

**>   Rabies,  diagnosis  of,  354. 

Rats,  white.  294. 

Ravenel's  method  of  making  agar,  16. 

on  diagnosis  of  rabies.  354. 
Ray  fungus.  263. 
Reaction  of  media,  8.  66. 
Ribbert's    method    of    concentrating    the 

tubercle  bacteria  in  sputum,  326. 
Rice  cooker,  6. 
Roll  cultures,  56. 
Rubber  dam  for  sealing  tubes.  20. 
Russell's   water  sampler,   128. 

OACCHAROMYCES  cerevisise,  50. 
a    Salt,  6. 

solution,  physiological,  18. 
Saprogenic  class.  91,  113. 
Saprophilic  class,  91,  95. 
Sarcina,  89. 

group.  91,  163. 

lutea,  40. 

tetragena,  163.  304,  314. 
Sections,  310,  312. 

Sedgwick's  method  of  air  analysis,  126. 
Shake  culture,   74. 
Slides,   cleaning  of,   26. 
Smegma  bacillus,  346. 
Spirillacese,  89. 
Spirillum,  90. 

cholerse  Asiaticse,  251. 

rubrum,   4. 
Spirochaeta.  89. 

Obermeieri,  332,  346. 
Spirosoma,  89. 
Spore  stain,  48. 
Spores,   study  of,   46. 
Sputum,  centrifugalizing,  328. 

examination  of  for  bacteria,  326. 

stain  of  for  tubercle  bacteria,  62,  326. 
Soapstone  for  cooling  plates,  54. 
Sodium  chloride,  6. 

hydrate,  8. 

nitrate  solution,  64. 

nitrite,  78. 
Soil  analysis,   132. 
Stage  micrometer.  36. 
Stain,  ahilin,  28. 

bottles.  30. 

Bunge's  for.  flagella,  48. 

Ernst's,  50. 

Gabbett's.  62. 

Gram's,  60. 

Hauser's,  48. 

Loeffler's,   312. 

solutions,  28. 

Weigert's,  314. 

Ziehl's,  326. 
Stab  cultures,  22,  85. 
Staphylococcus,  epidermidis  albus,  145. 

pyogenes  albus,  145. 

pyogenes  aureus,  149. 
Steam  pressure,  12. 

sterilizer,  10. 
Sterilization,  discontinuous,  10. 

of  glassware,  4. 


INDEX 


395 


Sterilization  of  instruments,  300. 

of  media,  10. 
Sterilizer,  hot  air,  4. 
Sternberg's  bulbs,  330. 
Streak  cultures.  24,  86. 
Streptococcus,  89. 

erysipelatos,  141,  304,  314,  348. 

lanceolatus,  307. 

pyogenes,  141. 
Streptothrix,  90. 

actinomyces,  263. 

bovis,  263,  314,  318,  330,  352. 

group,  92,  263. 

Madurse.  267. 

Sugar  meuia,  sterilization  of,  12. 
Sulphanilic  acid,  76. 
Sulphuric  acid,  30. 
Sulphuretted  hydrogen,  78. 
Sunlight,  effect  on  bacteria,  72, 
Swine  plague  group.  91,  183. 
Swine  erysipelas,   195. 
Syringe,  sterilization  of,  300. 

use  of,  294. 

^TAXONOMY,  82. 

-1-    Temperature,  effect  on  growth,  68. 

Testing  antiseptic  action,  134. 

disinfecting  action,   136. 
Test-tube,  cleaning  of,  2. 

cultures,  22. 


igar,  24. 
)lood 


blood  serum,  24. 
gelatin,  22. 
potato.  24. 
study  of,  26. 
staining  of,  30. 
Tetanus  group,  92,  289. 
Thermal  death  point,  68. 
Thermo-regulator,  12,  26. 
Thermostat,  24. 
Thionin,  344. 
Thiothrix,  90. 
Thrush,  organism  of,  324. 
Tin  foil,  for  sealing  tubes,  20. 
Tissue  embedding,  310. 
fixing,  310. 

handling  sections,  310. 
preparation  of  for  examination,  310. 
sectioning,  310. 


Tissue,  staining  of  bacteria  in,  312 
Titration  of  media,  6.  ' 
Transudates,  examination  of,  346. 
Tubercle  bacillus,  213. 

group,  92,  213. 

stain.  6^,  326. 
Tyndallization,  10. 
Typhoid  bacillus,  235. 

group,  92,  235. 

•JJRINE,  examination  of,  346. 

VIBRIO  cholerae,   251. 
v    Metschnikovi,  255. 
Schuylkilliensis,   259. 
subtilis,  99. 

Van  Gehucten  &  Xeli&'  method  of  diag- 
nosing rabies,  354. 

V\rATER  analysis,  128,  358. 

reaction  of  media  for,  8. 

bath,  56. 

blanks,   18. 
Weigert's  iodine  solution,  314. 

stain,  314. 

Welch's  capsule  stain,  50. 
Wertheim's  medium  for  the  gonococcus, 

158.  3*8. 
Wesbrook's  animal  cage,  300. 

hanging-drop  culture,  44. 
White  mice.  ^94. 

rats.  294. 

staphylococcus.  145. 
Widal  reaction.  334. 
Wright's  method  of  cultivating  anaerobes, 

Wurtz'  lactose  litmus  agar,  76. 
bacillus,   203. 


YEASTS>  morphology  of,  50. 


carbol-fuchsin,  28. 
Ziehl-Neelsen's  stain.  326. 
Zymogenic  class,  91,  109. 


Printed  in  the  United  States  of  America. 


XC  08529 


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UNIVERSITY  OF  CALIFORNIA  LIBRARY 


